| Objective:To study the clinical effect of Suyin detoxification prescription(SDP),and to analyze pharmacological mechanism through in vitro and in vivo experiments.Part Ⅰ:Retrospective cohort study which to compare the clinical efficacy of SDP and traditional syndrome differentiation prescription in the treatment of chronic kidney disease(CKD)non-dialysis patients at stages 3 to 5,deficiency of kidney,dampness and turbid syndrome.PartⅡ:To observe the effects of Suyin detoxification granule(SDG)on proteinuria,renal function,renal fibrosis and inflammation in rats induced by adenine.Part Ⅲ:The effects of SDP on albumin-induced mitochondrial dysfunction,cell pyroptosis and epithelial-mesenchymal transition(EMT)in renal tubular epithelial cells in vitro,and the molecular mechanism was further explained.Method:Part Ⅰ:Based on the Donghua Outpatient system,the CKD non-dialysis patients treated by SDP and traditional Chinese medicine syndrome differentiation prescription with renal function at stages 3 to 5,deficiency of kidney,dampness and turbid syndrome were screened in the outpatient department of nephrology of Jiangsu Province Hospital of Traditional Chinese Medicine from October 2017 to October 2020.The patients were divided into two groups:the control group(traditional syndrome differentiation prescription group)and the treatment group(SDP group)according to different treatment methods,and the baseline data of the patients were collected.The patients were followed up 6 months after treatment.The clinical data of the patients were recorded again,including the changes of proteinuria,serum albumin,serum urea nitrogen,serum creatinine,serum uric acid and glomerular filtration rate,the incidence of end point events,as well as the total effective rate of the two groups.Part Ⅱ:Adenine intragastric administration was used to establish renal failure rat model.The experiment was divided into four groups:blank group(normal saline intragastric administration),adenine group(2.5%adenine intragastric administration at 200 mg/kg/d for one month,and then continued to maintain the same dose of adenine intragastric administration alternate days for another one month,SDG low-dose group(adenine model+SDG 5 g/kg/d continuous intragastric administration for 2 months),and SDG high-dose group(adenine model+SDG 10 g/kg/d continuous intragastric administration for 2 months).Mental state,body weight and renal weight of the rats were recorded.After the rats were sacrificed at the end of the experiment,H&E,Masson and PAS stainings were used to observe the renal pathology.Blood urea nitrogen,serum creatinine,serum uric acid and serum cystatin C were used to evaluate renal function.Collagen Ⅰ and Fibronectin were detected by immunohistochemistry.Serum IL-1,IL-1β,IL-18 and MCP-1 were detected by Elisa to evaluate systemic inflammation.Renal inflammation related indexes(NLRP3,Caspase-1,Gasdermin-d,IL-lβ)were detected by immunohistochemistry.Part Ⅲ:HK-2 cells were stimulated by albumin to establish injury model.Cell activity was detected by CCK-8,cell migration ability was detected by cell scratch test,and the optimal concentration of SDP was selected to intervene the cell model.Western Blotting was used to explain the expressions of proteins like the epithelial-mesenchymal transition(EMT):Fibronectin,Collagen I,Vimentin,E-cadherin,and the pyroptosis pathway of NLRP3/ASC/Caspase-1/Gasdermin-D/IL-1β.The release of reactive oxygen species(ROS),the production of ATP,the content of superoxide dismutase(SOD)and the change of mitochondrial membrane potential were measured to evaluate the effect of SDP on the mitochondrial dysfunction induced by albumin.HK-2 cells were instantaneously transfected with Si RNA of mitochondrial antiviral protein(MAVS)to knock down the gene.Western Blotting was used to detect the expression of NLRP3 protein induced by albumin after Si MAVS knockdown.RT-PCR was used to detect the changes in mRNA contents of MAVS and NLRP3 after SDP,CY-09 and Si MAVS intervention in albumin model.The localization and quantitative expression of MAVS and NLRP3 induced by SDP,CY-09 and Si MAVS were further detected by immunofluorescence technique.Flow cytometry with Annexin V/FITC double channels was used to detect the effects of SDP,CY-09 and Si MAVS on cell pyroptosis.Result:Part Ⅰ:The baseline data of the control group(117 cases)and the treatment group(105 cases)were comparable.After 6 months of treatment,the two groups of patients were followed up.Statistically,the urinary protein in the treatment group was reduced compared with the control group(P=0.004),but the change of serum albumin was not obvious,40.79±5.47 g/L in the control group and 40.04±5.50 g/L in the treatment group(P=0.280).Compared with the control group,blood urea nitrogen in treatment group decreased significantly,which were 11.86±5.64 and 12.50±6.71 mmol/L(P=0.026).Compared with the control group,the serum creatinine in the treatment group decreased significantly(178.78±125.15 and 217.62±162.17 μmol/L,respectively)(P=0.029).There was no significant difference in serum uric acid between the two groups(P=0.099),which was 437.52±105.67 and 425.97±91.94 μmol/L respectively.Compared with the control group,the eGFR in SDP group increased significantly,which was 47.41±26.49 and 40.97±22.29 ml/min/1.73m2(P=0.039).Finally,the total effective rate was estimated by the serum creatinin,and the total effective rate was 74.29%in the treatment group versus 60.68%in the control group,which was statistically significant(P=0.031).The incidence of endpoint events was 2.86%in the treatment group and 5.13%in the control group,which was not statistically significant(P=0.392).Part Ⅱ:SDG can reduce adenine-induced renal weight gain,weight loss,and the expressions of Fibronectin and Collagen I,indicators of renal fibrosis,likewise improve the pathological results of renal fibrosis and inflammatory infiltration.At the same time,SDG decreased serum urea nitrogen,serum uric acid,serum creatinine,serum cystatin C and other indicators of renal function,decreased protienuria excretion rate,and increased serum albumin content.Secondly,serum inflammatory markers IL-1,IL-1β,IL-18 and MCP-1 were also decreased in SDG intervention.Finally,SDG reduced the expression of NLRP3,Caspase-1,Gasdermin-D,and IL-1β in kidney.Part Ⅲ:After SDP and CY-09 interfered with albumin model,cell activity increased,cell migration ability decreased,cell morphology changed from long fusiform to normal,protein expression of Fibronectin,Collagen I and Vimentin decreased,and protein expression of E-cadherin increased.NLRP3,ASC,activated Caspase-1,activated Gasdermin-D,and mature IL-1β protein expression were decreased.Meanwhile,SDP can improve mitochondrial dysfunction in cells,which is manifested in reduced ROS release,increased SOD and ATP content,and normal mitochondrial membrane potential.After knocking down MAYS gene,not only the MAVS protein expression was decreased,but NLRP3 protein expression was also decreased.Similarly,in the albumin model,both the proteins of MAVS and NLRP3 were decreased after Si MAVS intervention.After the intervention of SDP,CY-09 and SiMAVS,the mRNA levels of NLRP3 and MAVS were decreased.Immunofluorescence showed that MAVS co-located with mitochondria.In the albumin model,the co-localization of NLRP3 and MAVS was enhanced and the fluorescence expressions were enhanced.After the intervention of SDP,CY-09 and Si MAVS,the co-localization of NLRP3 and MAVS was weakened and the fluorescence expression intensity decreased.SDP,CY-09 and Si MAVS all reduced albumin-induced pyroptosis which were detected by flow cytometry.Conclusion:① SDP can reduce proteinuria,improve renal function and increase glomerular filtration rate in CKD non-dialysis patients with renal function at stages 3 to 5,deficiency of kidney,dampness and turbid syndrome.② SDG can relieve renal insufficiency,excess urinary protein,renal fibrosis and inflammation in adenine-rat model.③ SDP regulates mitochondrial dysfunction/MAVS/NLRP3-ASC-caspase-1 inflammatory pathway to reduce albumin-induced pyroptosis and EMT in renal tubular epithelial cells. |
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