| Objective To observe the difference of expression of microRNAs in rats with adriamycininduced renal fibrosis compared with normal rats,and exploring the effect of expression profile of microRNAs in rats with adriamycin-induced renal fibrosis after being treated with low、medium and high doses of Kangxianling decoction.At the same time to verify that different doses of Kangxianling decoction could improve renal fibrosis by inhibiting TGF-beta/Smad signaling pathway.Methods Sixty male Sprague-Dawley rats of SPF were divided into sham operation group,model group,group of Kangxianling decoction with low-dose or medium-dose or highdose,and Cossin group with stochastic.Model of renal fibrosis was established by cutting off right kidney and injecting with adriamycin into tail vein repeatedly.Rats were executed after eight weeks of treatment.Serum creatinine,blood urea nitrogen and 24-hour urinary protein were measured to quantify renal function.Observing histopathological changes of kidney by dyeing experiment with HE and Masson.Realtime PCR was used to detect the expression levels of TGF-beta 1,Smad3,Col1 alpha 1 and Col1 alpha 2 genes.miRNAs were sequenced with Illumina Hiseq2500,and then principal component analysis and cluster analysis were used to detect the correlation of the expression levels of microRNAs among rats in each group.The differentially expressed genes were analyzed and screened by using the criteria of gene expression multiple change greater than 1.5 and P value < 0.05.Results 1.Compared with the sham-operated group,the renal function of the model group decreased significantly,while Scr,Bun and 24 hUPro increased(P<0.05).Compared with the model group,group of Kangxianling decoction with low-dose or medium-dose or high-dose and Cossin group improved the renal function significantly,while Scr,Bun and 24 h UPro decreased significantly(P<0.05),the improvement effect of Kangxianling Decoction was dose-dependent.2.Under light microscope could see that the renal tissue structure of sham-operated group was basically normal;the glomerulus of model group was mostly moderately and severely focal segmental sclerosis,the tubules degenerated with granular and vacuolar,a large number of inflammatory cells infiltrated,interstitial fibrosis,and the degree of renal fibrosis was significantly higher than that of sham-operated group(P<0.05);the glomerulus of group of Kangxianling decoction with low-dose or medium-dose or high-dose and Cossin group showed mild and moderate focal segmental sclerosis,inflammatory cell infiltration and fibrosis were less severe in the model group than in the middle and high dose groups(P<0.05),but there was no significant difference in the low dose group(P>0.05).3.Real-time PCR results showed that the expression of TGF-β1,Smad3,Col1a1 and Col1a2 genes in kidney tissues of model group,group of Kangxianling decoction with low-dose or medium-dose or high-dose and Cossin group were significantly higher than those of sham operation group(P<0.05).Compared with the model group,the expression of group of Kangxianling decoction with low-dose or medium-dose or high-dose and Cossin group decreased,and Kangxianling decoction group gradually became significantly different with the increase of the dose(P < 0.05),but the low-dose group had no statistical significance(P > 0.05).4.Principal component analysis showed that the distance between medium-dose Kangxianling decoction group and Cossin group was the farthest,the other groups were in the middle position,and the distance between high-dose Kangxianling decoction group and sham-operation group was the closest.Cluster analysis showed that the sham-operated group was clustered,while group of Kangxianling decoction with low-dose or medium-dose or high-dose were clustered with the sham-operated group.5.Differentially expressed genes analysis showed that 21 microRNAs were differentially expressed in the sham-operated group compared with the model group,and six,eleven and eighteen microRNAs were differentially expressed in the low,medium and high dose Kangxianling decoction groups(P<0.05).The expression of microRNAs was up-regulated in the model group,including miR-21、miR-147、miR-542-3、miR-15b、miR-6216、miR-344a-2、miR-3085,but downregulated in high-dose Kangxianling decoction group.miR-107 was downregulated in the model group while up-regulated in high-does Kangxianling decoction group.Conclusion 1.Kangxianling decoction can significantly improve the renal function of rats with adriamycin-induced renal fibrosis,and inhibit the expression of collagen I gene by inhibiting the TGF-beta/Smad signaling pathway,finally to improve renal fibrosis,among them,high-dose Kangxianling decoction has the strongest effect.2.There exist differential expression of microRNAs in rats with adriamycin-induced renal fibrosis compared with normal rats.Kangxianling decoction can effect the expression of microRNAs,and the intervention effect of high dose Kangxianling decoction is the most obvious. |
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