| Objective: To investigate the effect and mechanism of qishengshu capsule in the treatment of diabetic nephropathy by observing the intervention effect of qishengshu Capsule on intestinal flora of diabetic nephropathy(DN)model rats and its correlation with inflammatory factors IL-6,MCP-1 and TGF-β1.Methods: 50 SPF male SD rats were randomly divided into three groups: blank control group,model group,high dose group,low dose group and valsartan group.In addition to the control group,the other groups were fed with high-fat and high-sugar diet for 4 weeks,then streptozotocin(STZ)was injected into abdominal cavity to establish diabetic nephropathy rats model.After the model was established successfully,the normal saline(0.01 ml / G.D)was given to the control group and the model group,the high dose(2.4G / kg.D)was given to the high kidney group,the low dose(1.2g / kg.D)was given to the low kidney group,and the Valsartan group was given to the Valsartan(0.034 g / kg.D)once a day for 8 weeks.To observe the general situation of the rats,before treatment,2 weeks,4 weeks and 8 weeks after treatment,weight was weighed,blood glucose was measured,urine microalbumin / creatinine(ACR)was measured before treatment and 2 weeks after treatment,and 24 hours urine protein was measured before treatment,4 weeks and 8 weeks after treatment.After 8 weeks of treatment,the fecal samples of rats were collected,and the distribution of community structure was compared by 16 sDNA sequencing.After the rats werekilled,the kidneys were taken,the histological changes of kidneys were observed by HE staining,the renal function was detected by blood samples,and the expression levels of IL-6,MCP-1 and TGF-β1 in serum were detected by ELISA.As there were 4 deaths in the model group during the course of disease,the sample size of statistical data was 6 in each group,with a total of 30 samples.Results: 1.Weight of rats: before treatment,compared with the control group,the weight f ats in other groups increased significantly(p < 0.05),there was no significant difference between the groups(p > 0.05).After treatment,compared with the control group,the weight of the model group and the Valsartan group decreased significantly at all time points(p < 0.05),the weight of the low kidney group and the high kidney group decreased at 4 and 8 weeks of treatment(p< 0.05);compared with the model group,the weight of the low kidney group and the Valsartan group increased significantly at 4 and 8 weeks of treatment(p < 0.05),and the weight of the high kidney group increased significantly at the beginning of the second week of treatment(p < 0.05);the treatment group Compared with the low kidney group,the weight of the high kidney group increased significantly(p < 0.05).Compared with the Valsartan group,the weight of the high kidney group increased significantly(p < 0.05).There was no significant difference between the low kidney group and the Valsartan group(p > 0.05).2.Blood glucose level of rats: compared with the control group,the blood glucose level of each model group at each time point was significantly higher(p< 0.05);the blood glucose level of high kidney group,low kidney group and valsartan group had no change before and after treatment(p> 0.05).3.Changes of 24-hour urinary protein quantity in rats: compared with the control group,the difference of 24-hour urinary protein quantity in other groups was not statistically significant(p> 0.05);compared with the control group,the level of 24-hour urinary protein quantity in other groups increased significantly(p< 0.05)in the fourth and eighth weeks of treatment,compared with the model group,the level of 24-hour urinary protein quantity in each treatment group was significantly higher in the fourth and eighth weeks of treatment Compared with valsartan group,the 24-hour urine protein level of low kidney group and high kidney group increased(p< 0.05).There was no significant difference between low kidney group and high kidney group(p >0.05).4.Changes of ACR in rats: compared with that before treatment,ACR in each model group increased significantly after 2 weeks of treatment(p< 0.05).There was no significant difference in the ACR level of each group before treatment(p>0.05).5.Change of renal function in rats: after 8 weeks of treatment,compared with the control group,the level of serum creatinine and urea nitrogen in the rest groups increased significantly(p< 0.05);compared with the model group,the level of serum creatinine and urea nitrogen in each treatment group decreased significantly(p< 0.05);compared with the treatment group,the level of serum creatinine and urea nitrogen in each treatment group increased significantly(p<0.05),The decrease of serum creatinine and urea nitrogen was the most obvious in valsartan group,followed by high kidney group and low kidney group(p<0.05).6.HE staining of renal tissue: there wasno abnormal change in the morphology and structure of glomerulus,renal tubules and renal interstitium in the control group.Inflammatory cell infiltration,mesangial cell proliferation,basement membrane thickening,tubular epithelial cell vacuolation,lumen expansion were observed in the model group.Basement membrane thickening and lumen expansion were significantly reduced in the Chinese medicine group and valsartan group.Renal injury degree: model group > valsartan group > kidney high group > kidney low group.7.Intestinal flora of rats: alpha diversity reflects the species diversity within a single sample.Measured by community richness index and community diversity index,the community richness index includes Chao1.The larger the index value is,the higher the community richness is;the community diversity index includes Shannon and Simpson,The larger the index,the higher the species diversity of the sample.Compared with the control group,the other groups’ Chao1 index decreased,but only the model group had statistical significance(p<0.05),Shannon and Simpson index decreased,but the difference was not statistically significant(p>0.05);compared with the model group,the number of Chao1 index in each drug treatment group increased,but only the Valsartan group had statistical significance(p<0.05).There was no significant difference in the indexes of Chao 1,Shannon and Simpson between the treatment groups(p>0.05).The relative abundance of phylum was dominated by Firmicutes,Bacteroides,Proteobacteria and actinobacteria.Compared with the control group,the abundance of Bacteroides increased and that of Spirillum decreased significantly(P.There was no significant differencebetween the two groups(p>0.05),but there was a trend of change.There was no significant difference between the two groups(p>0.05).Compared with the model group,the abundance of Bacteroides decreased in the high kidney group,increased in the low kidney group,and increased significantly in the Valsartan group(p<0.05),but there was no significant difference in the other three groups(p>0.05).Lactobacilli,Prevotella,blautia and alloprevotella are the dominant species.Compared with the control group,the lactobacilli abundance in the model group decreased significantly(p<0.05),and that in the high kidney group and the Valsartan group decreased significantly(p<0.05),but the difference was not statistically significant(p>0.05).Compared with the model group,the lactobacilli abundance of valsartan group decreased significantly(p<0.05),and the lactobacilli abundance of high kidney group and low kidney group increased,but the difference was not statistically significant(p>0.05).There was no significant decrease in Prevotella abundance in each treatment group(p>0.05).8.Changes of inflammatory factors in rats: compared with the control group,the levels of IL-6,TGF-β 1 and MCP-1 in the other groups were all increased(p<0.05);compared with the model group,the expression levels of IL-6,TGF-β 1 and MCP-1 in the low kidney group,the high kidney group and the Valsartan group were significantly decreased(p <0.05);compared with the Valsartan group,the expression levels of IL-6,TGF-β 1 and MCP-1 in the high kidney group and the low kidney group were significantly lower(p<0.05)There was no significant difference in IL-6,TGF-β 1 and MCP-1 between the two groups(p>0.05).9.Correlation analysis of intestinal flora andinflammatory factors: the flora abundance data does not meet the normal distribution,so using Spearman correlation coefficient statistical method,R is close to 1 or-1 to prove the higher correlation.If the species abundance is higher,the expression level of inflammatory factors is lower,indicating that there is a negative correlation between them.TGF-β 1 was negatively correlated with Chao1 index(p<0.05),IL-6,MCP-1 was negatively correlated with Chao1 index.At the level of phylum and genus,we can see that the inflammatory factors IL-6,MCP1,TGF-β have a negative correlation trend with sclereobacterium,proteus,Spirillum,Lactobacillus,etc.,and a certain positive correlation trend with actinomycetes,Bacteroides,blanket bacteria,etc.Conclusion1.Qishi Shenshu capsule can effectively reduce 24-hour urine protein quantity,serum creatinine and urea nitrogen level in DN rats,and delay the process of renal function damage.2.The species richness of DN rats was significantly lower than that of normal rats.Qishishenshu capsule may regulate the intestinal flora by improving the intestinal environment and maintaining the dominant number of beneficial bacteria.3.Qishishenshu capsule may protect renal function by down regulating the levels of IL-6,TGF-β1 and MCP-1.4.The correlation analysis of intestinal flora species richness and inflammatory factors IL-6,TGF-β 1,MCP-1 was less statistically significant,most of them had negative correlation trend. |
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