Effect Of Qingshen Granule On Inflammation Based On NLRP3 Signaling Pathway Inflammatory State Of Patients With Chronic Renal Failure And Intervention Mechanism Of HK-2 Cell Injury Induced By LPS

Objective: Observation of the expression of blood NLRP3 inflammatory vesicle-related molecules(NLRP3,ASC,Caspase-1,IL-1β,IL-18)in patients with Chronic renal failure(CRF)and the intervention effect of Qingshen granules.Methods: Sixty-eight sufferers with CRF damp-heat syndrome were included in the clinical trial,and they were randomly divided into treatment group and control group.each group has 34 cases,and 20 cases in the normal group.The study was finally completed in 63 cases(31 cases in the treatment group and 32 cases in the control group).During the study period,the treatment group stopped taking Qingshen Granules in 2 cases,plus Niaoduqing Granules in 1 case,and the control group stopped taking retention enema in 1 case,plus In 1 case of Shenshuaining Tablets,a total of 5 subjects were excluded.Both groups of patients were treated with blood pressure reduction,correction of anemia,regulation of calcium and phosphorus disorders,Chinese medicine retention enema and other treatments,and comorbidities were treated in a balanced manner.The treatment group took Qingshen Granules for 12 weeks,three times a day,10 g each time.After 12 weeks,observe Scr,e GFR,peripheral blood mononuclear cells NLRP3,ASC,Caspase-1 and Caspase-1 before and after treatment.The changes of IL-1β and IL-18 in serum and evaluation of curative effect.Result:1.Clinical efficacy: The total effective rates of the treatment group and the control group were 78.42% and 65.62%,the difference between the two groups was significant(P<0.05).2.TCM syndrome efficacy: The total effective rates of the treatment group and the control group were 80.65% and 59.37%,the difference between the two groups was prominently(P<0.05).3.TCM syndrome integral: Before therapy,there was inapparent difference between the two groups(P > 0.05);post-therapy,the syndrome integral values of the two groups decline(P < 0.01,P < 0.05),and the treatment group was obviously better than the control group(P < 0.01);there was no difference between the two groups(P > 0.05).4.Serum creatinine,estimated glomerular filtration rate(Scr,e GFR): There was no difference in renal function between the two groups before treatment(P>0.05);after 12 weeks of administration,the Scr level in the treatment group decreased(P<0.01),e GFR Increase(P<0.01),which is significantly different from the control group in the same period(P<0.01);there was no striking discrepancy in renal function in renal function before and after treatment in the control group(P>0.05).5.Comparison of NLRP3,ASC,Caspase-1 levels:5.1 Prior to treatment,the levels of NLRP3,ASC,and Caspase-1 in peripheral blood mononuclear cells of patients with CRF damp-heat syndrome were higher than those in the normal group(P<0.01);there was no discernible difference between the treatment group and the control group(P>0.05).5.2 After 12 weeks of administration,The levels of NLRP3,Caspase-1,and ASC in the two groups were lower than before(P<0.01,P<0.05),the treatment group was better than the control group(P<0.05).6.Comparison of IL-1β and IL-18 levels:6.1 Prior to treatment,the levels of serum IL-1βand IL-18 in normal group were lower than those in CRF patients with damp heat syndrome(P<0.01),there was no obvious distinction between the treatment group and the control group(P>0.05).6.2 After 12 weeks of administration,the levels of IL-1β and IL-18 in the serum of the two groups of tested patients were lower than before(P<0.01),the treatment group was better than the control group(P<0.01).7.Safety indicators: During this clinical study,there was no prominent change in the safety indicators of patients after 12 weeks of administrationConclusion:1.elevated levels of peripheral blood mononuclear cells NLRP3,ASC and Caspase-1 as well as serum IL-1β and IL-18 levels in patients with CRF moist-heat evidence.2.Qingshen Granules can down-regulate the levels of peripheral blood mononuclear cells NLRP3,ASC and Caspase-1 as well as serum IL-1β and IL-18 levels in patients with CRF damp-heat evidence,and reduce the inflammatory response.3.Qingshen granule reduces the clinical symptoms of CRF patients with damp-heat evidence,improve kidney function and inhibit the damage of inflammation to the body is one of its mechanisms;4.During this clinical study,Qingshen Granules had no adverse drug reactions,indicating that the drug is safe.Objective: To detect the human renal tubular epithelial cells induced by LPS in serum of Qingshen granules in different dosage groups HK-2 of NLRP3,ASC,caspase-1,IL-1β,IL-18 and α-SMA were studied.The mechanism of Qingshen granule was studied by inhibiting the inflammatory response mediated by NLRP3 signaling pathway,reducing the inflammatory damage of HK-2 and delaying the renal interstitial fibrosis(RIF).Methods: Eight healthy New Zealand male big-eared white rabbits weighing 2.5±0.2 kg were randomly divided into a drug group and a blank group,with 4 rabbits in each group.In the drug group,Qingshen granules were prepared into 1.0g/ml medicinal solution,and each rabbit was gavaged at 5ml/kg each time,once per day,for 10 consecutive days.One hour after the last gavage,under no anesthesia,blood was collected from the heart with a syringe and stored at-80°C for later use.Animals in the blank group were given the same amount of normal saline intragastrically to prepare blank serum.In the experiment,the 2-3 generation human renal tubular epithelial cells(HK-2)were selected,and the same generation cells were selected.They were randomly divided into normal control group,LPS group,NLRP3 inflammasome-specific inhibitor group(MCC950),Qingshen granule low,Middle and high dose groups.Observe cell morphology changes;CCK8 method to detect cell viability;Western blot method to detect NLRP3,ASC,Caspase-1,IL-1β,IL-18,and α-SMA protein expression levels in HK-2 cells;RT-q PCR,cellular immunofluorescence,flow cytometry were used to detect the expression of NLRP3,ASC,Caspase-1 and cell apoptosis in HK-2 cells.Result:1.The results of CCK8 showed that after 24 hours of drug intervention: Compared with the normal group,the cell OD values of other groups were strikingly lower than those of the normal group(P<0.01);compared with the LPS group,the OD of the NLRP3 inhibitor group and the Qingshen granule each dose group Both were increased(P<0.01);compared with the NLRP3 inhibitor group,the OD value of the Qingshen granule high and medium dose groups increased to varying degrees(P<0.05),but the Qingshen granule low-dose group and the NLRP3 inhibitor group,there was no striking discrepancy(P>0.05).Compared with the different dose groups of Qingshen Granules,the OD value of the Qingshen Granules high-dose group increased the most(P<0.05,P<0.01),and the Qingshen Granules middle-dose group was higher than the Qingshen Granules low-dose group(P< 0.05).Refer to the results of CCK8 to select 24 hours as the drug action time2.expression of NLRP3,ASC,caspase-1,IL-1β,IL-18,α-SMA in each group of cells(Western blot method)2.1 Compared with the normal group,the expression of NLRP3 protein in the cells of other groups increased to varying degrees(P<0.01);compared with the LPS group,the expression of NLRP3 protein in each dose group of Qingshen Granules and the NLRP3 inhibitor group were declined(P<0.01);compared with NLRP3 Compared with the inhibition group,the expression of NLRP3 protein in the Qingshen granule high and medium dose groups was significantly reduced(P<0.01),but there was no striking discrepancy between the Qingshen granule low-dose group and the NLRP3 inhibition group(P>0.05);each group of Qingshen granule In comparison,the expression of NLRP3 protein in the Qingshen granule high-dose group was the lowest(P<0.01),and the Qingshen granule middle-dose group was lower than the Qingshen granule low-dose group(P<0.01).2.2Compared with the normal group,the expression of ASC protein in the cells of other groups increased(P<0.01);compared with the LPS group,the expression of ASC protein in each dose of Qingshen Granules and the NLRP3 inhibitor group decreased to varying degrees(P<0.01);Compared with the NLRP3 inhibition group,the expression of ASC protein in each dose group of Qingshen Granulescut cut down varying degrees(P<0.01,P<0.05);among the dose groups of Qingshen Granules,Qingshen granule has the largest decrease(P<0.01),The low dose of Qingshen Granules decreased significantly less than that of the Qingshen Granules middle dose(P<0.05).2.2 Compared with the normal group,the expression of Caspase-1 protein in the cells of other groups increased to varying degrees(P<0.01);compared with the LPS group,the expression of Caspase-1 protein in each dose group of Qingshen Granules and the NLRP3 inhibitor group decreased to varying degrees(P<0.01,P<0.05);Compared with the NLRP3 inhibition group,the expression of Caspase-1 protein in each dose group of Qingshen Granules was obviously reduced(P<0.01,P<0.05);in each dose group of Qingshen Granules,Qingshen Granules The expression of Caspase-1 protein in the high-dose group was the lowest(P<0.01,P<0.05),and there there was no striking discrepancy between the middle and low-dose Qingshen granule groups(P>0.05).2.4 Compared with the normal group,The expression of IL-1β protein in each group increased to varying degrees(P < 0.01);compared with LPS group,the expression of IL-1 β protein in each dose group and NLRP3 inhibitor group was decreased(P < 0.01,P < 0.05);compared with NLRP3 inhibitor group,the expression level of IL-1β in high dose group of Qingshen granule decreased(P < 0.01),and the ratio of the middle and low dose group to NLRP3 inhibitor group was lower than that of the NLRP3 inhibitor group(P > 0.05),and the high dose group of Qingshen granule decreased most significantly(P < 0.01),and the middle dose group was lower than that of the low dose group(P < 0.05).2.5 Compared with the normal group,the expression of IL-1β protein in other groups was striking increased(P<0.01);compared with the LPS group,the expression of IL-1βprotein in each dose group of Qingshen Granules and the NLRP3 inhibitor group were all varying decreased(P<0.01,P<0.05);Compared with the NLRP3 inhibitor group,the expression level of IL-1βprotein in the Qingshen granule high-dose group was striking reduced(P<0.01).there was inapparent difference between the Qingshen granule medium and low-dose group and the NLRP3 inhibitor group(P>0.05);among the Qingshen granule groups,the largest decrease of IL-1βprotein in Qingshen granule high-dose group(P<0.01),and the Qingshen granule lower-dose group was higher than the Qingshen middle-dose granule group(P <0.05).2.6 Compared with the normal group,the expression of α-SMA protein in cells of other groups was are increased to varying degrees(P<0.01);compared with the LPS group,the expression of α-SMA protein in each dose group of Qingshen Granules and the NLRP3 inhibitor group shrink(P<0.01,P<0.05);Compared with the NLRP3 inhibitor group,the expression of α-SMA protein in each dose group of Qingshen Granules was reduced(P<0.01);comparison between the groups of Qingshen Granules,the largest decrease of α-SMA protein in Qingshen granule high-dose group(P<0.01),and the lower dose of Qingshen Granules was higher than that of the Qingshen Granules low-dose group(P<0.01).3.The expression of NLRP3,ASC and caspase-1 m RNA in each group(RT-qPCR method): Compared with the normal group,the expression of NLRP3,ASC and caspase-1mrna in each group was increased(P<0.01);Compared with LPS group,the expression of NLRP3,ASC and caspase-1mrna in each dose group and NLRP3 inhibitor group was decreased(P<0.01);Compared with NLRP3 inhibition group,the expression of NLRP3,ASC and caspase-1mrna in high and medium dose groups decreased(P<0.01),and there was no significant difference between the low dose group and the low dose group(P>0.05);The results showed that the above indexes decreased the most in the high dose group(P<0.01),and the middle dose group was lower than that of the low dose group(P<0.01).4.The expression of NLRP3,ASC,and Caspase-1 in each group of cells(immunofluorescence): NLRP3,ASC,and Caspase-1 were almost not expressed in the normal group,and almost no red fluorescence.In the LPS group,the expression was significantly enhanced,showing bright red fluorescence.In each dose group of Qingshen Granules and NLRP3 inhibitor group,the expression of red fluorescence was weakened to varying degrees compared with LPS group,and the expression of Qingshen Granules high-dose group was the weakest.5.Apoptosis in each group(flow cytometry):compared with the normal group,the apoptosis rate in each group was significantly increased(P<0.01);compared with the LPS group,the apoptosis rate in each dose group of Qingshen Granules and NLRP3 inhibitor group increased(P<0.01);compared with the NLRP3 inhibitor group,the apoptosis rate in each dose group of Qingshen Granules striking decreased(P<0.01);compared with each group of clear kidney granules Compared with the NLRP3 inhibitor group,the apoptosis rate was reduced in all dose groups(P<0.01).Conclusion1.LPS induced inflammatory damage,apoptosis and EMT in HK-2 cells.2.The serum containing Qingshen granule can inhibit the inflammatory damage and EMT process of HK-2 cells induced by LPS.3.The activation of NLRP3 inflammatory signaling pathway is involved in the inflammatory response and EMT of HK-2 cells.4.The serum containing Qingshen granule can reduce the expression of NLRP3,ASC,caspase-1,IL-1β,IL-18 and α-SMA in HK-2 cells induced by LPS by inhibiting the activation of NLRP3 inflammatory signaling pathway,thus inhibiting the inflammatory response,reducing the inflammatory damage and EMT of HK-2,and delaying the progress of renal fibrosis.5.The high dose group of Qingshen granule had better effect on the activation of NLRP3 inflammatory signal pathway,alleviating the inflammatory damage of HK-2cells and delaying renal fibrosis,which was better than that of the middle and low dose groups.