| 1.Objective To verify the clinical efficacy of Shendi Granules in patients with chronic glomerulonephritis(CGN)spleen and kidney deficiency syndrome by randomized controlled clinical trial,explore the mechanism of immune imbalance and the modulatory target of Shendi Granules in patients with chronic nephritis via observe the expression of key molecules in PD-1/PD-L1 pathway,and provide potential theoretical basics and guidance for the clinical application of Shendi Granules.2.Methods Sixty CGN spleen and kidney deficiency syndrome patients were included.All patients were randomly divided into: Valsartan group(n=20),Shendi Granules + Valsartan group(n=20),and Shendi Granules group(n=20).Patients in the Shendi Granules group were given Shendi Granules one packet each time(10g/bag),3 times a day for 12 weeks through warm water scouring;Shendi Granules + valsartan group were given Shendi Granules one packet each time,3 times a day for 12 weeks,meanwhile given valsartan once a day(morning service)for 12 weeks and 80 mg each time;Valsartan group were given valsartan once a day(80mg each time,morning service)for 12 weeks.During the administration of treatment,peripheral blood and urine samples of each group were collected for the detecting of 24-hour urine protein quantitation(24h Upro),ACR,TCR,renal function(Scr,BUN,e GFR),the activity of PD-1 and PD-L1,and the content of CD4+ and CD8+ T cell.Also,peripheral blood samples and urine samples of the control group were collected for the same detection as other groups mentioned.In addition,kidney tissues of 12 cases of membranous nephropathy,6 cases of focal segmental glomurular sclerosis,8 cases of minimal change nephropathy,6 cases of mesangial proliferative glomerulonephritis,2 cases of diabetic nephropathy,1 case of lupus nephritis,and 1 normal individual were collected for the detection of PD-1,PD-L1,CD4+,CD8+,and CD68.3.Results 3.1 Comparison of TCM syndrome scores: after the treatment,TCM syndrome scores of the valsartan group,the Shendi Granules group,and the Shendi Granules + valsartan group showed decline in comparison with before treatment(P<0.05).Compared TCM syndrome scores between each two groups,there had no statistically significant(P<0.05)between Shendi Granules group and the Shendi Granules + valsartan group;however,the Shendi Granules group shown better than the Valsartan group(P<0.05).3.2 Comparison of TCM Syndromes: the total effective rates of the Valsartan group,the Shendi Granules group,the Shendi Granules+Valsartan group were75%,90%,90%,respectively.Significant difference was observed between the Shendi Granules group and the Valsartan group(P<0.05),no significant difference was observed between the Shendi Granules group and the Shendi Granules+The Valsartan group(P>0.05),and there were significant differences between the Valsartan groups and the Shendi Granules+Valsartan groups(P<0.05).3.3 The total effective rates of the Valsartan group,the Shendi Granules group,and the Shendi Granules+Valsartan group were 60%,75%,and 90%,respectively.The differences were statistically significant(P<0.05).Significant difference was observed between the Shendi Granules group and the Valsartan group(P<0.05),the Shendi Granules group and the Shendi Granules+Valsartan group(P<0.05),and the Valsartan group and the Shendi Granules+Valsartan group(P<0.05).3.4 Comparison of the 24 h Upro,the ACR,and the TCR: compared with before treatment,there were significant differences in 24 h Upro,ACR,and TCR in the Valsartan group,the Shendi Granules group,and the Shendi Granules+Valsartan group at 4th week,the 8th week and the 12 th week(P<0.05),and the efficacy showed a time dependent manner.24 h Upro,ACR,and TCR were no significantly different between any two treatment groups at 4th week,but showed significantly different between any two treatment groups at 8th and 12 th week,which observed the effect of Shendi Granules+Valsartan was better than that of Shendi Granules(P<0.05)and Shendi Granules group was better than Valsartan group(P<0.05).3.5 The content of PD-1 and PD-L1 in PBMC: compared with the control group,the levels of PD-1 and PD-L1 in PBMC of the Valsartan group,the Shendi Granules group,and the Shendi Granules+Valsartan group were increased before the treatment,and no significant difference was observed among the three groups(P>0.05).The levels of PD-1 and PD-L1 in PBMC of the three groups were down-regulated(P<0.05)after treatment in comparison with the control group;Shendi Granules + Valsartan group showed lower than Shendi Granules group(P<0.05),and Shendi Granules group lowered than Valsartan group(P<0.05).3.6 CD4+,CD8+ T cell content,and CD4+/CD8+ ratio in PBMC: compared with the control group,the CD4+ T cell content in PBMC of the Valsartan group,the Shendi Granules group,and the Shendi Granules+Valsartan group were decreased before treatment;the CD8+ T cell content was increased in PBMC of the Valsartan group,the Shendi Granules group,and the Shendi Granules+Valsartan group before treatment;the CD4+/CD8+ ratio of the Valsartan group,the Shendi Granules group,and the Shendi Granules+Valsartan group were decreased before treatment;significant difference observed between the comparisons(P<0.05).There were no significant differences in CD4+ T cell content,CD8+ T cell content,and CD4+/CD8+ ratio among the Valsartan group,the Shendi Granules group,and the Shendi Granules+Valsartan group(P>0.05).The CD4+T cell content in PBMC of the Valsartan group,the Shendi Granules group,and the Shendi Granules+Valsartan group were increased after treatment,the CD8+ T cell content was decreased in PBMC of the Valsartan group,the Shendi Granules group,and the Shendi Granules+Valsartan group after treatment,the CD4+/CD8+ ratio of the Valsartan group,the Shendi Granules group,and the Shendi Granules+Valsartan group were increased after treatment,and significant difference observed between the comparisons(P<0.05).The effect of Shendi Granule+Valsartan was better than that of Shendi Granule(P<0.05),and the effect of Shendi Granule was better than that of Valsartan(P<0.05).3.7 Expression of PD-1,PD-L1,CD4+,CD8+,and CD68 proteins in kidney tissues: the expressions of PD-1,PD-L1,CD4+,CD8+,and CD68 were detected by immunofluorescence and Western blot and two methods showed consistent results;all the proteins observed different degrees of expression.The PD-1,PD-L1,CD8+,and CD68 had the lowest expression in normal individual kidney tissues and highest expression in nodular diabetic nephropathy;CD4+ had the highest expression in normal individual kidney tissues and the lowest expression in nodular diabetic nephropathy.3.9 Renal function: the renal function indicators(Scr,BUN,e GFR)were improved among the Shendi Granules group,the valsartan group,the Shendi Granules+valsartan group and within the normal range.3.10 Safety indicators: there were no significant changes in safety indicators before and after treatment among Shendi Granules group,Valsartan group,Shendi Granules+ Valsartan group,and no adverse drug reactions were observed during treatment period.4.Conclusion 4.1 Shendi Granules could effectively improve the clinical symptoms of patients with CGN(spleen and kidney deficiency syndrome),and significantly reduce the level of urinary protein.The effect of Shendi Granules+Valsartan was the most significant,followed by Shendi Granules and Valsartan.4.2 In the PBMC of patients with CGN spleen and kidney deficiency syndrome,CD4+T cell was down-regulated,CD8+T expression was up-regulated,and the key molecule of the costimulatory signaling pathway PD-1/PD-L1 was positively expressed.Shendi Granules may intervene in the key molecules of the PD-1/PD-L1 signaling pathway,thereby regulating the T cell activity to maintain moderate immune function and protect renal function.4.3 Different pathological types of glomerular disease,compared with normal individuals,the expression of PD-1,PD-L1,CD8+,and CD68 were significantly increased.In the primary glomerular diseases,the mesangial proliferative glomerulonephritis had expressed lowest,and the minimal change nephropathy expressed the highest.Among the secondary glomerular diseases,the lupus nephritis expressed the lowest,and the diabetic nephropathy expressed the highest.Compared with normal individuals,the expression of CD4+ was decreased;in primary glomerular disease,mesangial proliferative glomerulonephritis had the highest expression,and minimal change nephropathy had the lowest expression;in secondary glomerular disease,the lupus nephritis had the highest expression and the diabetic nephropathy had the lowest expression.1.Objective To explore the mechanism of Shendi Granules in the regulation of co-stimulatory signaling molecule PD-1/PD-L pathway to interfere the immune imbalance of chronic glomerulonephritis patients by observing and analyzing the pathological changes of renal tissue,the content of the CD4+ and CD8+ T-lymphocytes in PBMC,and the expression of PD-1 and PD-L1 protein,which in the renal tissue of rat model of mesangial proliferative glomerulonephritis(Ms PGN).2.Methods Forty male sprague dawley rats(2 months old and weighing 200±20g)were purchased from Experimental Animal Center of Anhui Medical University.After a week of adaptive feeding,all rats were randomly divided into control group,model group,Shendi Granules group,and valsartan group;there are ten rats in each group.Except the normal group,all the rats of the other groups were injected with Thy-1 antibody via by tail vein injection to establish the anti-Thy-1 mesangial proliferative nephritis rat model.Four weeks after the antibody injection,the rats of the Shendi Granules group were garaged with aqueous solution of Shendi Granules(4.0 g/kg·d),the rats of valsartan group were given suspension of valsartan capsules(20 mg/kg·d),and the rats of the other groups were garaged an equal volume of water.All rats were administrated for 12 weeks,and 24-hour urine was collected via a metabolic cage during the period of administration.At the end of the administration,blood samples were taken from the heart for the detection of the expression of CD4+ and CD8+ T cell content in the PBMC,rat kidneys tissues were used to detect the expression of PD-1 and PD-L1 protein,and the observation of renal histopathological changes.3.Results 3.1 Rats of the control group of had a good mental state,normal diet,and shiny hair.While,rats of the other groups showed a poor mental state and diet,and the hair was scorched.After administrated with Shendi Granules and valsartan,the mental state of the rats were gradually improved,and it was close to rats in the control group.3.2 Urinary protein changes: Compared with the control group,levels of 24 h Upro of the other groups were significantly increased after modeling(P<0.05=.The 24 h Upro levels of the valsartan group and the Shendi Granules group were decreased(P<0.05),and the Shendi Granules group decreased significantly in comparison with the valsartan group.3.3 The expression of CD4+ and CD8+ in the peripheral blood,changes of the CD4+/CD8+ ratio,and the expression of key molecules in the PD-1/PD-L pathway: Compared with the control group,the count of CD4+ was decreased(P<0.05),the count of CD8+ was increased in the peripheral blood(P<0.05),changes of CD4+/CD8+ ratio was decreased(P<0.05),and the expression of PD-1 and PD-L1 in renal tissue were increased.Compared with the model group,both the Shendi Granules group and the valsartan group shown increased in the expression of CD4+(P< 0.05),decreased in the expression of CD8+(P< 0.05),increased in the changes of CD4+/CD8+(P< 0.05),and the expression of PD-1 and PD-L1 in renal tissue were decreased(P< 0.05).The effects of Shendi Granules were better than that of valsartan.3.4 The glomerular mesangial cell proliferation and mesangial matrix increase in the Shendi Granules group and the valsartan group were improved in comparison with the model group,and the Shendi Granules was superior than the valsartan.4.Conclusion 4.1 Shendi Granules could effectively reduce the level of urine protein of Ms PGN rats and improve the quality of life.4.2 There had immunological damage in the kidney of Ms PGN rats,resulted in decreased of CD4+ content in the peripheral blood,increased of CD8+ content in the peripheral blood,and increased expression of PD-1 and PD-L1 in renal tissue.4.3 Shendi Granules may regulate T cell activity,which via up-regulating the expression of CD4+ T cells,decreasing the expression of CD8+T and down-regulating the expression of PD-1 and PD-L1 in PD-1/PD-L1 pathway in PBMC,to maintain moderate immune function in the body and improve proliferation of mesangial cells and stroma in Ms PGN rats. |
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