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Back to Journal »Journal of Inflammation Research» Volume 14

Chinese medicine Suyin Jiedu Granules inhibits pyrolysis and epithelial-mesenchymal transition by down-regulating MAVS/NLRP3 to reduce kidney injury

Authors: Zhu Yan, Huang Kun, Yang Yan, Yong C, Yu Xin, Wang Gang, Yi Li, Gao Ke, Tian Fei, Qian Sheng, Zhou E, Zou Yan

Published on December 7, 2021, the 2021 volume: 14 pages 6601-6618

DOI https://doi.org/10.2147/JIR.S341598

Single anonymous peer review

Reviewing editor: Professor Quan Ning

Zhu Yiye,1,2 Huang Guoshun,1,2 Yang Yang,1,2 Chen Yong,1,2 Xiang Yu,1,2 Wang Gang, 3 Lan Yi, 1,4 Kungao, 1,4 Fangtian, 2 Qian Shushu ,2 Zhou Enchao,1,4 Zou Yanqin 1,3,4 1 Department of Nephrology, Jiangsu Provincial Hospital of Traditional Chinese Medicine, Affiliated Hospital of Nanjing University of Traditional Chinese Medicine, Nanjing, Jiangsu; No. 2 1 Clinical School of Nanjing University of Traditional Chinese Medicine, Nanjing, Jiangsu; 3 Zou's Nephropathy Medicine Intangible Cultural Heritage Inheritance Studio, Nanjing Broad Kidney Disease Hospital, Nanjing, Jiangsu; 4 Master Zou Yanqin Inheritance Studio, Jiangsu Traditional Chinese Medicine Hospital, Nanjing, Jiangsu Correspondent: Zhou Enchao; Zou Yanqin Tel: +86-13851572603; +86-25-86538830 Fax +86-25-86538830 Email [email protection]; [email protection] Purpose: Proteinuria is an independent risk factor for chronic kidney disease (CKD). By activating the NLRP3 inflammasome, albumin-induced tubulointerstitial inflammation and epithelial-mesenchymal transition (EMT) are potential therapeutic targets for CKD. Suyin Jiedu Granules (SDG) improve proteinuria and delay renal failure. However, the underlying mechanism is still unknown. Methods: Firstly, a rat renal failure model was established by gavage with adenine. Assess renal function, proteinuria, serum inflammation indicators and renal pathology, and perform renal immunohistochemical staining on NLRP3 inflammasome after intervention of low and high concentration SDG. Secondly, the renal tubular epithelial HK-2 cell model was established with albumin in vitro, and the cell viability, EMT phenotype and the expression of NLRP3 inflammasome signaling pathway protein (SDP) and CY-09 after freeze-dried powder of Suyin Jiedu Recipe were measured. A selective and direct NLRP3 inhibitor, co-incubated with albumin. ATP, SOD, mitochondrial membrane potential, and ROS were further measured in vitro to observe the changes of mitochondrial function after SDP intervention. Use siRNA to knock down the mitochondrial antiviral signal protein (MAVS), and use Western blot, polymerase chain reaction (PCR) and immunofluorescence to verify the interaction between MAVS and NLRP3. Results: SDG improved renal function and proteinuria, reduced renal fibrosis, reduced serum inflammation and the expression of NLRP3 inflammasome components in the kidney. In vitro, SDP and CY-09 enhanced the cell viability after albumin injury, and inhibited the pyrolysis induced by the NLRP3 inflammatory signaling pathway and the expression of proteins involved in EMT. It was further found that SDP alleviated the mitochondrial dysfunction caused by albumin. Knockdown of MAVS reduces the expression of NLRP3 pathway proteins and their mRNA levels, and also weakens the co-localization of NLRP3, thereby reducing pyrolysis. Conclusion: SDP protects renal tubular epithelial cells from pyrolysis and EMT by regulating albumin-induced mitochondrial dysfunction/MAVS/NLRP3-ASC-caspase-1 inflammasome signaling pathway. Keywords: Suyin Jiedu Granules, renal tubular epithelial cells, mitochondrial dysfunction, MAVS, NLRP3 inflammasome, pyrolysis, EMT

Chronic kidney disease (CKD) is a growing public health problem. The global prevalence of CKD is 8%-16%. 1 Because renal failure is progressive and irreversible, and lack of specific drugs to protect renal function, the quality of life of CKD patients is threatened and burdens their families. 2 It is a clinician and researcher to reduce renal tissue damage and renal dysfunction. The huge challenges faced by personnel. 3

In the process of renal fibrosis, endogenous renal cells are damaged due to pathogenic factors, leading to increased collagen deposition, leading to gradual hardening of the renal parenchyma, scarring, and loss of basic metabolic functions, leading to end-stage renal disease (ESKD). 4 Renal fibers The changes are characterized by persistent inflammation, the increase and aggregation of myofibroblasts, and abnormal deposition of extracellular matrix (ECM). More and more evidences indicate that epithelial-mesenchymal transition (EMT) plays an important role in tubulointerstitial fibrosis. 5,6 During EMT, cell morphology changes, cell migration increases, adhesion factors E-cadherin and tight junction protein ZO-1 decrease, and extracellular matrix proteins, such as fibronectin and type I collagen, increase. 7

In recent years, inflammasome research has become more and more popular. Among the identified inflammasomes, the NLRP3 inflammasome has been extensively studied. 8,9 NLRP3 inflammasome integrates infectious and non-infectious tissue damage signals. The most classic way is to activate caspase-1, which induces the secretion of pro-inflammatory cytokines IL-1β and IL-18 and inflammatory cell death (pyrolysis), which promotes the host's defense against pathogens and tissue repair. 10 Existing data indicate that in addition to renal mononuclear phagocytes, certain intrinsic renal cells, such as renal tubular epithelial cells, podocytes, glomerular endothelial cells, and mesangial cells, also contain large amounts of NLRP3. 11 In several CKD In disease models, the release of Il-1β and the activation of NLRP3 inflammasomes are associated with kidney inflammation, EMT and fibrosis. 12-14 Therefore, the NLRP3 inflammasome is a potential therapeutic target for CKD.

After more than 2500 years of continuous development, traditional Chinese medicine has gradually become a clear treatment system, which is widely used in the treatment of diseases. Suyin Jiedu Recipe (SDP) promotes blood circulation, dissipates heat, nourishes the kidney and detoxifies. SDP has been used to treat chronic renal failure for many years. In our previous research, it was found that SDP's prince drugs, Perilla leaf [Perilla leaf] and Yinchen [Artemisia annua], have antioxidant and anti-apoptotic effects. 15 However, the mechanism of SDP's renal protective function is still unclear. Adenine causes renal failure and proteinuria in rodents, which is a model for studying CKD. 16 We used an adenine rat renal failure model to explore the effects of SDP on urine protein, renal function, and inflammation indicators. In vitro, the use of albumin to damage human renal tubular epithelial HK-2 cells to simulate the use of proteinuria to destroy renal tubules, to explore the effect of SDP on the mitochondrial dysfunction of MAVS activation of NLRP3, and to study its induction effect in HK-2 cells Jiao death and EMT.

Adenine (A8626) was purchased from Sigma-Aldrich (MO, USA). Albumin Cohn Fraction V (4240GR100) from bovine serum (BSA) was purchased from BioFROXX (Germany). CY-09 (HY-103666) was purchased from MedChemExpress (NJ, USA). SDG was purchased from Jiangsu Provincial Hospital of Traditional Chinese Medicine (2005001, Jiangsu, CN). It is currently an in-hospital preparation of Jiangsu Provincial Hospital of Traditional Chinese Medicine, which is sold by the hospital. Antibodies against type I collagen (67288-1-lg), fibronectin (66042-1-lg) and MAVS (14341-1-AP) were purchased from Proteintech (Chicago, USA). The primary antibody against Cleaved IL-1β (AF4008) was purchased from Affinity Biosciences (OH, USA). The primary antibody against pro Caspase-1 + p10 + p12 (ab179515) was purchased from Abcam (Cambridge, UK). The primary antibody of Gasdermin D (N-terminal; ER1901-37) was purchased from HUABIO (Zhejiang, China). The primary antibody against NLRP3 (NBP2-12446) was purchased from Novus (CO, USA). Mito-Tracker Red CMXRos (C1035) was purchased from Beyotime (Shanghai, CN). HRP conjugated goat anti-rabbit IgG (H + L) and HRP conjugated goat anti-mouse IgG (H + L) were purchased from Servicebio (Hubei, China). CoraLite488 conjugated Affinipure goat anti-rabbit IgG (H + L) and CoraLite594 conjugated Affinipure goat anti-rabbit IgG (H + L) were purchased from Biosharp (Shanghai, China).

SDP uses Perilla leaf [Folium Perillae], Silver Chen [Herba Artemisiae Scopariae], Liu Yuexue [Serissa japonica Thunb.], Tu Fu Lin [Smilax glabra Roxb.], Safflower [Carthamus tinctorius L.], Wu to prepare Lingzhi [Trogopterori], Pu Huang [Typha angustifolia L.], Rheum Officinale [Rheum Officinale], Muli [Concha Ostreae], Poria [Poria], Astragalus [Radix Astragali], Mangosteen [Cornus officinalis] Xibo]. All medicinal materials come from Jiangsu Provincial Hospital of Traditional Chinese Medicine. Then, in the Institute of Pharmacology of Nanjing University of Traditional Chinese Medicine, the quick-drinking detoxification freeze-dried powder was prepared. Apigenin (B20981), rhein (B20245), aloe-emodin (B20772), formononetin (B20836) and ferulic acid (B20007) were purchased from Yuanye Biotechnology Company (Shanghai, China). 4ʹ-hydroxyacetophenone (BCBV5820) was purchased from Sigma-Aldrich (St. Louis, USA). Chlorogenic acid (HY-N0055), quercetin (HY-18085) and scopolamine (HY-N0228) were purchased from MedChemExpress (NJ, USA). We use liquid chromatography tandem mass spectrometry (LC-MS/MS) to analyze SDP. After separation on an Xbridge C18 column (2.1 × 100 mm; 3.5 µm; Waters, USA) using an acetonitrile-water gradient system, the peaks were analyzed using ESI ionization mass spectrometry (MS) in MRM mode. Use LC-MS/MS related software to collect and process data (Table 1 and Figure S1). Table 1 LC-MS/MS determination of SDP monomer components of traditional Chinese medicine

Table 1 LC-MS/MS determination of SDP monomer components of traditional Chinese medicine

Male SD rats (n = 24; 12 weeks old; body weight, 200-230 g) were purchased from Shandong Jinan Yueshi Animal Breeding Center (Shandong, China). After one week of adaptive feeding, the rats were given adenine by gavage. They were randomly divided into four groups (n = 6 in each group): control, adenine (Ade), SDG low-dose (SDG-L) and SDG high-dose (SDG-H) groups. In the Ade group, adenine (2.5%, 200 mg/kg/d) was administered intragastrically every day for 4 weeks, and then every other day for 4 weeks to maintain disease progression. The SDG high-dose group was given 10 g/kg/d SDG, and the low-dose group was given 5 g/kg/d SDG. From the second week, no more than 2 mL of compound solution was administered per day until the end of the experiment.

All animal experiments are in compliance with the guidelines for care and use of laboratory animals, and have been approved by the Animal Ethics Committee of the Affiliated Hospital of Nanjing University of Traditional Chinese Medicine (license number: 2020DW-27-02).

A fully automatic biochemical analyzer (Dimension EXL200, Siemens healthineers, USA) was used to determine blood urea nitrogen, serum creatinine, serum uric acid, serum cystatin and albumin. Urine analyzer (Uritest-500B, URIT, CN) was used to detect urine ACR. ELISA kit (Meimian, CN) was used to measure serum IL-1, IL-1β, IL-18 and MCP-1. All procedures were performed according to the instructions of the respective manufacturers.

The kidney tissues were fixed with 10% formalin and the specimens were taken, and the specimens were dehydrated by pressurized alcohol, transparent, immersed, embedded, sectioned, conventional H&E staining, Masson and Pas staining. An optical microscope (Eclipse Ni-U, Nikon, Japan) was used to observe histopathological changes. ImageJ is used to measure Masson's stained area.

After the paraffin sections are dehydrated, they are placed in a box containing EDTA antigen retrieval buffer (pH 9.0) and placed in a microwave oven for antigen retrieval (medium heat for 8 minutes to boiling, stop fire for 8 minutes, then medium heat for 7 minutes). After natural cooling, it was washed with PBS. Place the slides in 3% hydrogen peroxide solution and incubate for 25 minutes at room temperature in the dark. After blocking with 3% bovine serum albumin for 30 minutes at room temperature, the sample was incubated with the primary antibody (diluted in PBS) at 4°C overnight. Then the slides were washed with PBS and incubated with HRP-conjugated secondary antibody goat anti-rabbit/mouse IgG (H + L) for 1 hour at room temperature. After drying, add freshly prepared 3,3ʹ-diaminobenzidine color reagent (DAB staining solution, Servicebio, CN) to the labeled tissue. Use an optical microscope (Eclipse Ni-U, Nikon, Japan) to observe the glass slide until the cell nucleus turns yellow-brown, and then counter-stain with hematoxylin staining solution. Finally, the sample was dehydrated, clarified in xylene for 5 minutes, and sealed with a resin mounting agent. Use an optical microscope to observe the tissue.

Normal human renal tubular epithelial HK-2 cell line was purchased from Cellcook Biotechnology Co, LTD (Guangzhou, China), and obtained the STR gene identification certificate. The cells were grown at 37°C in Dulbecco's Modified Eagle's medium/F-12 (DMEM/F12) containing 10% FBS (GIBCO, USA) in an incubator containing 5% CO2.

Use CCK-8 kit (Dojindo, China) to measure cell viability. The cells are incubated in a 96-well plate (10,000 cells per well) with 100 μL medium containing 5% FBS per well. After drug stimulation, the medium was removed, and 100 μL of medium containing 10% CCK-8 detection solution was added to each well. After incubating for 1 hour, use a spectrometer to measure the absorbance at 450 nm. Cell viability is expressed as a percentage of the control.

Mark vertical lines on the outer surface of each hole of the 6-well plate. The cells were seeded in a 6-well plate at a density of 200,000 cells per well. After the cells are fused to about 90%, the medium is removed. A thin "wound" perpendicular to the marking line is introduced by scraping the cells with the pipette tip. The cells were gently washed twice with PBS, and each drug was diluted in serum-free medium, and then added to a 6-well plate. After the inspection, the image was taken using an optical microscope. ImageJ is used to measure the scratched area.

Sample preparation: After treatment, cells were lysed with a solution containing protease and phosphatase inhibitor (Beyotime, CN). After lysis, the lysate was collected and sonicated and centrifuged. Collect the supernatant, add SDS loading buffer, and boil at 100°C for 10 minutes. The samples were then subjected to SDS-PAGE electrophoresis (Bio-RAD, China) on 10% or 12.5% ​​gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membrane was blocked with 5% skimmed milk powder in PBST (PBS containing 1‰ Tween-20) for 1 hour at room temperature, the primary antibody was incubated overnight at 4°C, washed with PBST, and the secondary antibody was incubated for 1 hour, and then washed again. Use the imaging system to visualize the bands and analyze them with Image Lab software.

The cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature, and then permeabilized with 0.1% Triton X-100 in PBS. After blocking with 3% bovine serum albumin in PBS, the sample was incubated with the primary antibody (diluted 1:200) at 4°C overnight. Then, the sample was incubated with CoraLite488-conjugated Affinipure goat anti-rabbit IgG (H + L) (1:200 dilution) for 1 hour at room temperature. After the sample is gently shaken to dry, drop CY3-TSA (Tyramide signal amplification) into the circle, and incubate for 10 minutes at room temperature in the dark. Add another primary antibody (1:200 dilution) and incubate the sample overnight at 4 °C. After washing with PBS, the sample was incubated with CoraLite594-conjugated goat anti-rabbit IgG (H + L) (1:200 dilution) for 1 hour at room temperature. After washing with PBS, the samples were incubated with TSA for 10 minutes at room temperature and stained with DAPI staining solution for 15 minutes. Add anti-fluorescence quencher. Use a fluorescence microscope to observe cells using different channels. ImageJ is used to measure relative fluorescence intensity.

Use the one-step RNA extraction method (Vazyme, CN) to extract RNA, and use Direct RT Mix to synthesize cDNA according to the manufacturer's instructions. Use Direct qPCR MIX-SYBR to detect and amplify cDNA. The primers used are listed in the table below (Table 2). The 7500Fast system detects mRNA expression, and the results are calculated using the ΔΔCT method. The mRNA level is expressed as the score of the control group. Table 2 Primer sequence

JC-1 kit (Multi Sciences, Hangzhou, China) was used to detect mitochondrial membrane potential. After stimulation with the drug, the sample was gently washed with PBS, then trypsin without EDTA was added, and the sample was incubated at room temperature for 10 minutes. The cells are then collected and centrifuged. Discard the supernatant and resuspend the cells in a buffer containing 2 μM JC-1. The samples were incubated for 20 minutes at 37°C in a 5% CO2 incubator. Flow cytometry was performed using an excitation wavelength of 488 nm, and samples were analyzed using FITC and R-PE channels. Annexin V-FITC/PI Apoptosis Kit (Multi Sciences, China) was used to measure cell pyrolysis. After collection, the cells in each sample were incubated with 5 μL FITC and 2 μL PI. Annexin V-FITC and PI detection channels are used to measure cell pyrolysis.

HK-2 cells were transiently transfected with siRNA specifically targeting MAVS. siMAVS and sicontrol (negative reference) were provided by Nanjing KGI Biotech Co., Ltd. and were diluted in Opti medium to a final concentration of 80 nM. The transfection reagent used was Lipo 3000 (Thermo Fisher, USA). The siRNA and transfection reagent are mixed, incubated for 15 minutes, and then added to the cells. After culturing for 8 hours, the medium was removed. The transfected cells are then exposed to different stimuli.

After lysing the cells, collect 20 μL of the supernatant in an opaque 96-well plate. Then immediately add the ATP detection reagent. Use a fluorescence microplate reader to measure fluorescence and calculate ATP and protein concentrations. The ATP concentration is converted to nmol/mg. SOD: The supernatant of the cell lysate is used to measure the SOD level. After performing the experiment according to the manufacturer's instructions, use a spectrometer to detect the OD value at a wavelength of 450 nm. Calculate the SOD activity and protein concentration according to the instructions (S0101M, beyotime, Shanghai, CN), and convert the SOD activity to U/mg. ROS detection: After administration, add 500μL DCFH-DA fluorescein to each well according to the instructions (S0033M, Beyotime, CN), the final concentration is 10μM, and incubate at 37°C for 30 minutes. After sealing the sample with anti-fluorescence quenching solution, use a forward fluorescence microscope to directly observe the sample and take an image. Use ImageJ to measure relative optical density.

Values ​​are expressed as mean ± standard deviation. Before performing further statistical analysis, use the Shapiro-Wilk test to check whether the data is normally distributed. Student's t-test was used to compare the two populations. In order to compare between multiple samples, a one-way analysis of variance is performed first, and then Dunnett's test is performed. All analyses were performed using SPSS Statistics 23.0 software. A P value of less than 0.05 is considered to indicate a statistically significant difference.

The rat model of adenine nephropathy not only caused renal failure, but also appeared high proteinuria. It is a rat model of chronic renal failure. The general examination showed that compared with the control group, the adenine group lost weight, the kidney weight increased, the kidney was obviously swollen, white in color, and granular, and these effects were all weakened. Low-dose and high-dose SDG groups (Figure 1A and B). The results of renal immunohistochemistry (Figure 1C) showed that fibrosis markers collagen I and fibronectin in the adenine group were significantly increased, while their levels were decreased in the SDG low-dose and high-dose groups. H&E staining of renal cortex showed that compared with the control group, most of the renal interstitium in the adenine group was inflamed, the number of renal tubules was reduced, the structure of the kidney atrophied, and lymphocytes increased. Interstitial infiltration, enlargement of the renal tubule lumen around the lesion, and edema of epithelial cells. Masson staining tissue cortex is highly damaged, and collagen fibers proliferate more in the injured site and surrounding renal tubule interstitium, which is dark blue. PAS staining showed no obvious increase in mesangial matrix and thickening of basement membrane. However, a small number of renal tubules in the medulla were PAS-positive, and these lesions were improved in the low-dose and high-dose SDG groups (Figure 1D). These results indicate that SDG reduces kidney damage caused by adenine. Figure 1 SDG improves renal fibrosis in rats with adenine nephropathy. Experimental schedule: Adenine, SDG-L and SDG-H groups were injected with adenine (2.5%, 200 mg/kg/d) daily for 4 weeks, and then for another 4 weeks every other day. SDG-L and SDG-H groups were given 5 g/kg/d or 10 g/kg/d SDG by gavage once a day. The rats were sacrificed at the 9th week. At this time, (A) observe the morphology of the kidney, (B) measure the body weight and kidney weight, (C) and use immunohistology to evaluate the representative image of the kidney section using collagen I (Col I) and fibronectin (FN) Antibody. Scale bar = 50 µm. (D) Hematoxylin and eosin staining: In the adenine group, extensive renal interstitial inflammation (brown arrow) was observed. The number of renal tubules is reduced (black arrow), and renal atrophy and structural changes (blue arrow) are observed. There is increased lymphocyte infiltration in the interstitium (yellow arrow), and part of the renal tubule lumen around the lesion is enlarged; in addition, the epithelial cells are swollen (red arrow). Masson staining: In the adenine group, the cortex was extensively damaged, and there were more collagen fibers in the dark blue area (black arrow) around the damaged site and the tubulointerstitium. Periodic acid-Schiff staining: There was no significant increase in the mesangial matrix and thickening of the basement membrane in the adenine group. However, a few tubules in the medulla are PAS positive (black arrow). Scale bar = 50 µm. Data are expressed as mean ± SD; n=6. ##P <0.01 and control. *P <0.05; ** P <0.01 compared with the adenine group.

Figure 1 SDG improves renal fibrosis in rats with adenine nephropathy. Experimental schedule: Adenine, SDG-L and SDG-H groups were injected with adenine (2.5%, 200 mg/kg/d) daily for 4 weeks, and then for another 4 weeks every other day. SDG-L and SDG-H groups were given 5 g/kg/d or 10 g/kg/d SDG by gavage once a day. The rats were sacrificed at the 9th week. At this time, (A) observe the morphology of the kidney, (B) measure the body weight and kidney weight, (C) and use immunohistology to evaluate the representative image of the kidney section using collagen I (Col I) and fibronectin (FN) Antibody. Scale bar = 50 µm. (D) Hematoxylin and eosin staining: In the adenine group, extensive renal interstitial inflammation (brown arrow) was observed. The number of renal tubules is reduced (black arrow), and renal atrophy and structural changes (blue arrow) are observed. There is increased lymphocyte infiltration in the interstitium (yellow arrow), and part of the renal tubule lumen around the lesion is enlarged; in addition, the epithelial cells are swollen (red arrow). Masson staining: In the adenine group, the cortex was extensively damaged, and there were more collagen fibers in the dark blue area (black arrow) around the damaged site and the tubulointerstitium. Periodic acid-Schiff staining: There was no significant increase in the mesangial matrix and thickening of the basement membrane in the adenine group. However, the few tubules in the medulla are PAS positive (black arrow). Scale bar = 50 µm. Data are expressed as mean ± SD; n=6. ##P <0.01 and control. *P <0.05; ** P <0.01 compared with the adenine group.

As shown in Figures 2A and B, after adenine treatment, rats' blood urea nitrogen, serum creatinine, serum cystatin, and blood uric acid increased, and urine albumin excretion rate and serum albumin decreased. The low-dose and high-dose SDG groups showed improvements in renal function and hypoalbuminemia, and decreased urinary protein excretion. Compared with the control group, the serum inflammatory markers IL-1, IL-1β, IL-18 and MCP-1 in the adenine group were significantly increased, and the inflammation in the low-dose and high-dose SDG groups was improved (Figure 2C). NLRP3/ The caspase-1/IL-1β signaling pathway regulates inflammation through the lysis and maturation of pro-inflammatory cytokines, and has been shown to play a role in regulating kidney inflammation. As shown in Figure 2D, adenine increased NLRP3, caspase-1, GSDMD, and IL-1β in kidney tissue, and low and high SDG doses reduced their levels. These data indicate that SDG improves adenine-induced renal dysfunction, proteinuria, hypoalbuminemia and inflammation, and inhibits the expression of NLRP3, caspase-1, GSDMD and IL-1β in the kidney. Figure 2 SDG alleviates adenine-induced decline in renal function and inflammation in rats. (A) Blood urea nitrogen (BUN), serum creatinine (Scr), serum cystatin, serum uric acid, (B) serum albumin, urine albumin to creatinine ratio (ACR) and (C) serum IL-1, IL The levels of -1β, IL-18 and MCP-1 were measured. (D) Representative immunohistological images of NLRP3, Caspase-1, Gasdermin D (GSDMD) and IL-1β using the respective antibodies. Scale bar = 50 µm. Data are expressed as mean ± SD; n=6. ##P <0.01 and control. *P <0.05; ** P <0.01 compared with the adenine group.

Figure 2 SDG alleviates adenine-induced decline in renal function and inflammation in rats. (A) Blood urea nitrogen (BUN), serum creatinine (Scr), serum cystatin, serum uric acid, (B) serum albumin, urine albumin to creatinine ratio (ACR) and (C) serum IL-1, IL The levels of -1β, IL-18 and MCP-1 were measured. (D) Representative immunohistological images of NLRP3, Caspase-1, Gasdermin D (GSDMD) and IL-1β using the respective antibodies. Scale bar = 50 µm. Data are expressed as mean ± SD; n=6. ##P <0.01 and control. *P <0.05; ** P <0.01 compared with the adenine group.

More and more studies have shown that proteinuria is an independent risk factor for renal failure. In order to study the effect of excess albumin on the kidney, especially the function of renal tubules, we treated renal tubular epithelial cells with serum albumin and established an injury model. As shown in Figure 3A, as the concentration of BSA increases and the intervention time increases, cell viability is affected. Compared with the control group, the 5 g/L SDP low-dose group showed no difference at 12 and 48 h, but increased at 24 h, while the 10 g/L and 20 g medium and high-dose groups/L SDP were at 24 and 48 h, respectively. Hours show loss of cell viability. The scratch test results show that 5 g/L, 10 g/L and 20 g/L BSA can improve cell migration at 12 and 24 hours. Observed by light microscope, 24 hours after the intervention, the cell morphology of the BSA group changed from ellipse to spindle, and the cell volume increased. BSA at a concentration of 10 g/L showed the most significant change (Figure 3B and C). Western blot results (Figure 3D) showed that the levels of vimentin, collagen I, and fibronectin in the BSA 10 g/L group increased, while the levels of E-cadherin decreased. In order to verify whether albumin caused pyrolysis of renal tubular epithelial cells, the protein levels of NLRP3, ASC, activated caspase-1, GSDMD-N and activated IL-1β were measured, and they were found to increase in different concentrations after treatment with BSA ( Figure 3E). The most significant increase was found in the group administered with 10 g/L BSA. Western blot analysis showed that as the levels of activated caspase-1 and IL-1β increased, the levels of unactivated caspase-1 and IL-1β precursors also increased. These results indicate that albumin can be used to establish a renal tubular injury model with reduced function, enhanced migration ability, and increased expression of fibrosis marker protein and NLRP3 series of pyrode-related proteins. Figure 3 Renal tubular epithelial cell damage induced by albumin in vitro. (A) The effect of BSA on cell viability. Expose HK-2 cells in 96-well plates to different concentrations (0, 5, 10, and 20 g/L) of BSA for 12, 24, and 48 hours. The CCK-8 assay was used to assess cell viability. (B, C) The effect of BSA on cell migration and related morphological changes. Use a phase contrast microscope (magnification, 100X) for cell morphology and scratch determination. (D) The effect of BSA on the protein expression of vimentin, E-cadherin (E-cad), fibronectin (FN) and type I collagen (Col I). After 24 hours of incubation, cell lysates were subjected to Western blotting to measure vimentin, E-cadherin, fibronectin, and type I collagen levels. (E) The effect of BSA on NLRP3 inflammasome protein. After 24 hours of incubation, Western blot was performed on the cell lysate to measure NLRP3, ASC, Pro Caspase-1 with lytic caspase-1 levels, N-terminal of Gasdermin D (GSDMD-N), and Pro IL-1β with lytic IL -1β. Data are expressed as mean±SD; n=3. #P <0.05; ##P <0.01 and control.

Figure 3 Renal tubular epithelial cell damage induced by albumin in vitro. (A) The effect of BSA on cell viability. Expose HK-2 cells in 96-well plates to different concentrations (0, 5, 10, and 20 g/L) of BSA for 12, 24, and 48 hours. The CCK-8 assay was used to assess cell viability. (B, C) The effect of BSA on cell migration and related morphological changes. Use a phase contrast microscope (magnification, 100X) for cell morphology and scratch determination. (D) The effect of BSA on the protein expression of vimentin, E-cadherin (E-cad), fibronectin (FN) and type I collagen (Col I). After 24 hours of incubation, cell lysates were subjected to Western blotting to measure vimentin, E-cadherin, fibronectin, and type I collagen levels. (E) The effect of BSA on NLRP3 inflammasome protein. After 24 hours of incubation, Western blot was performed on the cell lysate to measure NLRP3, ASC, Pro Caspase-1 with lytic caspase-1 levels, N-terminal of Gasdermin D (GSDMD-N), and Pro IL-1β with lytic IL -1β. Data are expressed as mean±SD; n=3. #P <0.05; ##P <0.01 and control.

Based on the above results, we determined that 10 g/L BSA and 24 hours of intervention time are the best conditions for establishing a cell injury model. In order to study the relationship between NLRP3 inflammasome and EMT, the NLRP3 inhibitor CY-09 was used. CY-09 is a selective and direct NLRP3 inhibitor. It directly binds to the ATP binding motif of NLRP3 NACHT domain and inhibits NLRP3 ATPase activity, thereby inhibiting the assembly and activation of NLRP3 inflammasome. In order to study the protective effect of SDP, a model of BSA-induced kidney injury was used to incubate CY-09 (5 μM) with BSA and SDP (0.25, 0.5, and 1 g/L). The results showed that SDP (1 g/L) and CY-09 improved cell viability. CY-09 and SDP (0.5 and 1 g/L) improved the cell morphology and abnormal cell migration caused by BSA. The effect of 1 g/L SDP is more obvious (Figure 4A-C). The concentration of 1 g/L SDP was determined as the optimal concentration. Western blot results showed that SDP and CY-09 reduced the protein levels of vimentin, collagen I and fibronectin, and increased the protein levels of E-cadherin (Figure 4D). As shown in Figure 5A-C, SDP and CY-09 reduced the expression of NLRP3, thereby inhibiting the activation of the NLRP3-ASC-Caspase-1 pyrolysis pathway, reducing the activation of Gasdermin D and the release of IL-1β ( Figure 5). However, compared to SDP or CY-09, the combination of SDP and CY-09 does not appear to show differences in this BSA-induced EMT and pyroptosis cell model (Figure S2). These data indicate that SDP may improve renal tubular EMT by inhibiting NLRP3-mediated pyrolysis. Figure 4 SDP may improve the cell damage induced by BSA by inhibiting the NLRP3/ASC/caspase-1 signaling pathway. (A) The effect of SDP and CY-09 on cell viability induced by BSA. Incubate HK-2 cells in a 96-well plate with SDP (0.25, 0.5, and 1.0 g/L) or CY-09 (5 μmol/L) and BSA (10 g/L) for 24 hours. The CCK-8 assay was used to assess cell viability. (B, C) The effects of SDP and CY-09 on cell migration and morphological changes induced by BSA. Use a phase contrast microscope (magnification, 100X) to analyze cell morphology and scratch determination. (D) The effect of SDP and CY-09 on the expression of E-cadherin, vimentin, fibronectin and type I collagen. HK-2 cells are divided into 6 groups: Control, BSA (10 g/L), SDP (1 g/L), BSA (10 g/L) + SDP (1 g/L), CY-09 (5 μmol / L) and BSA (10 g/L) + CY-09 (5 μmol/L). After 24 hours of incubation, cell lysates were subjected to Western blotting to measure E-cadherin, vimentin, fibronectin, and type I collagen levels. Data are expressed as mean ± SD; n=3. #P <0.05; ##P <0.01 and control. *P <0.05; ** P <0.01 compared with BSA group. Figure 5 SDP may improve BSA-induced cell damage by inhibiting the NLRP3/ASC/caspase-1 signaling pathway. (AC) The effect of SDP and CY-09 on the expression of NLRP3 inflammasome protein. After 24 hours of incubation, Western blot was performed on the cell lysate to measure NLRP3, ASC, Pro Caspase-1 with lytic caspase-1, N-terminal of Gasdermin D (GSDMD-N), and Pro IL-1β with lytic IL- 1β data is expressed as mean ± SD; n=3. ##P <0.01 and control. ** P <0.01 compared with BSA group.

Figure 4 SDP may improve the cell damage induced by BSA by inhibiting the NLRP3/ASC/caspase-1 signaling pathway. (A) The effect of SDP and CY-09 on cell viability induced by BSA. Incubate HK-2 cells in a 96-well plate with SDP (0.25, 0.5, and 1.0 g/L) or CY-09 (5 μmol/L) and BSA (10 g/L) for 24 hours. The CCK-8 assay was used to assess cell viability. (B, C) The effects of SDP and CY-09 on cell migration and morphological changes induced by BSA. Use a phase contrast microscope (magnification, 100X) to analyze cell morphology and scratch determination. (D) The effect of SDP and CY-09 on the expression of E-cadherin, vimentin, fibronectin and type I collagen. HK-2 cells are divided into 6 groups: Control, BSA (10 g/L), SDP (1 g/L), BSA (10 g/L) + SDP (1 g/L), CY-09 (5 μmol / L) and BSA (10 g/L) + CY-09 (5 μmol/L). After 24 hours of incubation, cell lysates were subjected to Western blotting to measure E-cadherin, vimentin, fibronectin, and type I collagen levels. Data are expressed as mean ± SD; n=3. #P <0.05; ##P <0.01 and control. *P <0.05; ** P <0.01 compared with BSA group.

Figure 5 SDP may improve BSA-induced cell damage by inhibiting the NLRP3/ASC/caspase-1 signaling pathway. (AC) The effect of SDP and CY-09 on the expression of NLRP3 inflammasome protein. After 24 hours of incubation, Western blot was performed on the cell lysate to measure NLRP3, ASC, Pro Caspase-1 with lytic caspase-1, N-terminal of Gasdermin D (GSDMD-N), and Pro IL-1β with lytic IL- 1β data are expressed as mean ± SD; n = 3. ##P <0.01 and control. ** P <0.01 compared with BSA group.

Mitochondria not only produce the energy required by the cell, but also participate in the cell regulation process. Recently, studies have shown that they also play a role in regulating immunity. Previous studies have confirmed that mitochondrial dysfunction is one of the processes leading to the activation of NLRP3. As shown in Figure 6A-E, compared with the BSA group, the SDP group showed reduced ATP release and ROS production, and increased mitochondrial membrane potential and SOD release. These results may indicate that SDP can reduce the activation of NLRP3 inflammasomes by reducing mitochondrial dysfunction. Figure 6 SDP improves mitochondrial dysfunction induced by BSA. (A) The effect of SDP on the release of adenosine triphosphate (ATP) from cells induced by BSA. After 24 hours of incubation, use this kit to perform ATP determination on cell lysates. Use a fluorescence microplate reader to measure the ATP in the sample. (B) The effect of SDP on the changes in mitochondrial membrane potential (MMP) induced by BSA. JC-1 detection kit is used to measure MMP. The FITC channel is used to count cells damaged by mitochondria. (C) The effect of SDP on the release of superoxide dismutase (SOD) induced by BSA. The SOD released was measured using an SOD determination kit. (D, E) The effect of SDP on the production of reactive oxygen species (ROS) induced by BSA. The corresponding kit is used to measure ROS. Use a fluorescence microscope (magnification, 100X) to observe the cells. Data are expressed as mean±SD; n=3. ##, P <0.01 compared with the control. ** P <0.01 compared with BSA group.

Figure 6 SDP improves mitochondrial dysfunction induced by BSA. (A) The effect of SDP on the release of adenosine triphosphate (ATP) from cells induced by BSA. After 24 hours of incubation, use this kit to perform ATP determination on cell lysates. Use a fluorescence microplate reader to measure the ATP in the sample. (B) The effect of SDP on the changes in mitochondrial membrane potential (MMP) induced by BSA. JC-1 detection kit is used to measure MMP. The FITC channel is used to count cells damaged by mitochondria. (C) The effect of SDP on the release of superoxide dismutase (SOD) induced by BSA. The SOD released was measured using an SOD determination kit. (D, E) The effect of SDP on the production of reactive oxygen species (ROS) induced by BSA. The corresponding kit is used to measure ROS. Use a fluorescence microscope (magnification, 100X) to observe the cells. Data are expressed as mean±SD; n=3. ##, P <0.01 compared with the control. ** P <0.01 compared with BSA group.

MAVS localizes to the outer mitochondrial membrane, promotes localization of NLRP3 to the mitochondria and induces its activation. As shown in Figure 7A, the expression of NLRP3 was also reduced after the MAVS gene was knocked out. Compared with the BSA group, the siMAVS and SDP groups showed decreased expression of MAVS, suppression of NLRP3, and decreased mRNA content of MAVS and NLRP3 (Figure 7A-C). Immunofluorescence results showed that MAVS co-localized with mitochondria, and BSA enhanced the co-localization of NLRP3 and MAVS and increased fluorescence. SDP and siMAVS reduced their colocalization and fluorescence (Figure 7D). Interestingly, the CY-09 group showed decreased expression of NLRP3 and MAVS fluorescence, indicating that NLRP3 may regulate MAVS through positive feedback. Perform flow cytometry to detect the positive rate of Annexin V and PI double stained cells using channels PE and 488. Compared with the BSA group, the SDP, CY-09, and siMAVS groups showed a reduction in pyrolysis as measured by flow cytometry (Figure 7E). These data indicate that SDP inhibits the activation of NLRP3 inflammasomes by regulating the expression of MAVS, thereby reducing pyrolysis. Figure 7 SDP inhibits the localization and activation of NLRP3 in mitochondria by regulating the MAVS protein. (A, B) The effect of MAVS siRNA and SDP on cell damage induced by BSA. HK-2 cells were transfected with NLRP3 siRNA or control siRNA for 8 hours. Incubate the transfected cells with BSA for 24 hours. HK-2 cells are divided into 6 groups-Control, BSA (10 g/L), SDP (1 g/L), BSA (10 g/L) + SDP (1 g/L), siMAVS (80 nmol/L) ) And BSA (10 g/L) + MAVS (80 nmol/L). Western blot was performed on cell lysates to measure MAVS and NLRP3 levels. (C) The mRNA levels of NLRP3 and MAVS were measured using RT-PCR. (D) Use immunofluorescence staining to observe the co-localization of MAVS and NLRP3. (E) Annexin V -FITC and PI channels are used to observe cell pyrolysis. Data are expressed as mean±SD; n=3. #P <0.05; ##P <0.01 and control. *P <0.05; ** P <0.01 compared with BSA group.

Figure 7 SDP inhibits the localization and activation of NLRP3 in mitochondria by regulating the MAVS protein. (A, B) The effect of MAVS siRNA and SDP on cell damage induced by BSA. HK-2 cells were transfected with NLRP3 siRNA or control siRNA for 8 hours. Incubate the transfected cells with BSA for 24 hours. HK-2 cells are divided into 6 groups-Control, BSA (10 g/L), SDP (1 g/L), BSA (10 g/L) + SDP (1 g/L), siMAVS (80 nmol/L) ) And BSA (10 g/L) + MAVS (80 nmol/L). Western blot was performed on cell lysates to measure MAVS and NLRP3 levels. (C) The mRNA levels of NLRP3 and MAVS were measured using RT-PCR. (D) Use immunofluorescence staining to observe the co-localization of MAVS and NLRP3. (E) Annexin V -FITC and PI channels are used to observe cell pyrolysis. Data are expressed as mean±SD; n=3. #P <0.05; ##P <0.01 and control. *P <0.05; ** P <0.01 compared with BSA group.

Traditional Chinese medicine has a history of more than 2500 years in China. With the development of science and technology, several Chinese herbal medicines and their active ingredients have been proven to be effective in treating CKD. 17-19 However, following the principle of matching traditional Chinese medicines, the combination of drugs has better efficacy than single drugs, and the clinical application of traditional Chinese medicines is still based on Chinese herbal compound prescriptions. 20,21 SDP has been used clinically for many years with satisfactory results. For convenience, it has been converted to particles (SDG). This study is the first to explore the renal protective effect of SDP on adenine-induced renal failure in rats. The results show that SDP can not only improve kidney function and fibrosis, but also reduce proteinuria and inflammation. In vitro, the renal tubular epithelial cell model established using albumin indicated that SDP may reduce pyrolysis by reducing mitochondrial dysfunction, inhibiting the MAVS/NLRP3 inflammasome pathway, and further inhibiting the EMT of renal tubular epithelial cells (Figure 8). Figure 8 Schematic diagram of the signaling pathway involving SDP-mediated inhibition of BSA in human renal tubular epithelial cells (HK-2 cells).

Figure 8 Schematic diagram of the signaling pathway involving SDP-mediated inhibition of BSA in human renal tubular epithelial cells (HK-2 cells).

Many clinical trials have shown that proteinuria is an independent predictor of CKD progression. ESKD and the risk of cardiovascular mortality are positively correlated with proteinuria. 22 The protein in the initial urine is filtered by the glomerulus and reabsorbed from the renal tubules into the blood circulatory system. Proteinuria may be caused by immune and/or non-immune factors that impair glomerular filtration or damage renal tubules. 23 Proteinuria is a driving factor of renal tubulointerstitial inflammation and fibrosis. 24 However, the exact mechanism of proteinuria is unclear. The animal model of kidney injury is established by gavage of adenine, and adenine is deposited in the renal tubules under the action of xanthine oxidase. 16 Compared with other rodent models of renal failure established by intravenous or surgical methods, adenine gavage helps to strictly control the dose of the drug, reduce the mortality rate, and avoid the inflammatory interference caused by trauma. Our data showed that after taking 2.5% adenine continuously for 1 month and taking 200 mg/kg/d daily for 1 month, serum urea nitrogen, serum creatinine, serum uric acid and serum cystatin C increased, and observed To proteinuria, in line with the clinical manifestations of CKD. Pathological staining and immunohistochemistry also confirmed damage to renal tubules, including atrophy, loss of renal interstitial structure, inflammation and fibrosis. Pathological staining showed no serious glomerular damage, and PAS staining showed no obvious glomerular mesangial hyperplasia. We focused our attention on the renal tubules. Both low-dose (5 g/kg/day) and high-dose (10 g/kg/day) of SDG can alleviate the injury, and the effect of the high-dose group is more significant.

The mechanism of proteinuria leading to tubulointerstitial damage is an active area of ​​research. The currently known damage mechanisms include the direct toxic effects of proteins, oxidative stress, chemical additives, inflammasomes, cell apoptosis, cell senescence and autophagy. 25 In our previous study, the NLRP3 inflammasome was activated in response to proteinuria induced by doxorubicin nephropathy. 26 The assembly of NLRP3 inflammasome includes sensor-NLRP3, adaptor-ACS and progenase-procaspase-1. When stimulated by pathogen-associated molecular patterns (PAMP) and damage-associated molecular patterns (DAMP), inflammasomes act as polymer protein complexes to cause self-cleavage of caspase-1, leading to the conversion of IL-1β and IL-18 into mature IL-1β and IL-18 are released by cells and trigger an inflammatory response. At the same time, mature caspase-1 promotes Gasdermin D to form pores in the cell membrane, further enhancing the release of mature IL-1β and IL-18. This process is called pyrolysis. In addition to the classic caspase-1.27,28, it can also be induced by non-classical caspase-4, caspase-5 and caspase-11. As a kind of programmed inflammatory necrosis cell death, moderate pyrolysis can maintain a balance but excessive pyrolysis leads to the release of a large number of pro-inflammatory factors and inflammatory response. At present, it is known that pyrexia is related to the occurrence and development of infections, autoimmune diseases, neurodegenerative diseases, metabolic disorders and other diseases. 29 In the current study, NLRP3, Caspase-1, Gasdermin D and IL-measure 1β in kidney tissue and macrophage chemokines MCP-1, IL-1, IL-1β and IL-18 in blood. It is confirmed that the kidney damage caused by adenine is related to a series of reactions caused by the NLRP3 inflammasome. Experimental evidence shows that low-dose and high-dose SDG can attenuate these inflammatory responses, but the underlying mechanism is still unclear.

In addition to providing energy for cell activities, mitochondria are also involved in regulating cell functions, including stabilizing internal calcium concentration, cell apoptosis, cell signal transduction, and aging. 30 The involvement of mitochondria in immune regulation is an important finding. 31 There is evidence that mitochondria activate NLRP3 inflammasomes through three pathways. 32 – ① Mitochondria activate NLRP3 inflammasomes by releasing ROS: When mitochondria are dysfunctional, they will produce excessive ROS. Its clearance limit, excessive ROS will activate related signal transduction pathways and cause cell damage. Previous experiments have shown that mitochondrial dysfunction is an early event of podocyte and renal tubular epithelial cell damage. Blocking mitochondrial reactive oxygen species can reduce cell damage, suggesting that mitochondrial dysfunction plays an important role in the initial stage of cell damage. 33, 34 ② Mitochondrial DNA activates NLRP3 inflammasome: mtDNA is the genetic material in mitochondria. When mitochondrial dysfunction occurs, mtDNA is released into the cytoplasm, becomes DAMP and binds to NLRP3. 35 In order to further study the effect of mtDNA on the activation of NLRP3 inflammasomes, Nakahira et al.36 overexpressed mtDNA in bone marrow-derived macrophages (BMDMs) cells and found that the overexpressed mtDNA induced the activation of NLRP3 inflammasomes in a dose-dependent manner. Activation and expression of IL-1β. This indicates that the mtDNA released in the cytoplasm directly binds and activates the NLRP3 inflammasome, leading to mitochondrial dysfunction. ③ Mitochondrial co-localization with NLRP3 is essential for NLRP3 inflammasome activation: Toll-like receptors (TLR) and RIG-1 related to the plasma membrane and introns are recruited by the mitochondrial outer membrane protein MAVS to activate type I interferon The reaction is during the viral infection. Initially, it was thought that the assembly of activated NLRP3 inflammasomes in the cytoplasm might not be recruited in mitochondria. However, Subramanian et al. 37 demonstrated that NLRP3 is mainly located in the endoplasmic reticulum in the resting state, while NLRP3 and ASC are redistributed to the endoplasmic reticulum and mitochondria in clusters around the nucleus through NLRP3 inflammasome activator. Studies have further shown that the MAVS protein is necessary for the optimal activity of the NLRP3 inflammasome. MAVS interacts with the N-terminus of NLRP3 during inflammasome activation. Therefore, MAVS not only mediates the antiviral type I interferon response, but also acts as a bridge for mitochondria to regulate the NLRP3 inflammasome. 38 In addition to MAVS, apoptosis inhibitor protein (c-FLIP), 39 acetylated α-tubulin, 40 cardiolipin 41 on mitochondria also regulate NLRP3 mitochondrial localization.

In this study, by measuring mitochondrial function, it was observed that albumin affects the release of ATP, the level of mitochondrial membrane potential, the content of SOD and the release of ROS, indicating that albumin is in HK-2 cells and SDP can alleviate dysfunction. In the past, MAVS protein was thought to activate immune-related signaling pathways and induce the expression of interferon to participate in the antiviral immune response. 37 It has also been reported that MAVS regulates NLRP3 by relocating NLRP3 in the early stages of hypoxia. There is evidence that the MAVS protein expressed on the mitochondria is stimulated by albumin to increase its expression. At the same time, the co-localization of NLRP3 and MAVS increased as their expression increased. When siRNA was used to knock down MAVS, the co-localization and expression of MAVS and NLRP3 were weakened. Similar results were also found in the SDP group. It is worth noting that the NLRP3 inhibitor CY-09 also inhibited the MAVS protein. It is speculated that there is a complex interaction between NLRP3 and CY-09, rather than a direct causal relationship.

In summary, this study shows that in a rat model of adenine-induced kidney injury, SDP can improve proteinuria and renal fibrosis, reduce inflammation, and protect renal function. In addition, we explored the regulation of NLRP3 by MAVS induced by mitochondrial dysfunction, which involves inflammation-induced pyrolysis and renal tubular epithelial EMT, which may be a new target for the treatment of CKD. Our results indicate that SDP can protect renal tubular epithelial cells from pyrolysis and EMT by regulating the albumin-induced mitochondrial dysfunction/MAVS/NLRP3-ASC-caspase-1 inflammasome signaling pathway.

National Natural Science Foundation of China (81873270), Jiangsu Provincial Hospital of Traditional Chinese Medicine Science Project (Y2018RC17), Jiangsu Top Talent Training Program (WSN-012), Jiangsu Provincial Superior Discipline Project Funding Nanjing University of Traditional Chinese Medicine (ZYX03KF076), Nanjing Science and Technology Bureau Medical International Cooperative project (202002051).

The author said that he did not violate his rights.

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