| Objective:1.To observe the effect of Fushen decoction on peritoneal injury in uremic rats with peritoneal fibrosis(peritoneal fibrosis,PF).2.To explore the effect of Fushen decoction on the process of PF and its mechanism.Methods:110 healthy male SD rats were randomly divided into sham operation group(Sham group)and PF group.The PF group was divided into model group,benazepril group,low-dose Fushen decoction group and high-dose Fushen decoction group.The PF group was given intraperitoneal injection of 4.25% hyperglycemic peritoneal dialysate 20 ml 2 weeks after nephrectomy,lipopolysaccharide(LPS)(0.6mg/kg)was injected intraperitoneally on the 1st,2nd,4th and 8th day,and 62500 units of erythromycin were added on the 7th,14 th,21st and28 th day of the model.Each group was given different groups of drugs for 6 weeks from the beginning of intraperitoneal injection.The control group and model group were infused with3 ml saline.At the end of the 6th week of drug intervention,all the experimental animals were killed and the peritoneal tissue of rats was taken.Part of the peritoneal tissue was fixed in 4%paraformaldehyde,and the rest was preserved in liquid nitrogen for follow-up detection.The contents of serum creatinine(Scr)and blood urea nitrogen(BUN)were detected by serology.The morphological changes of peritoneal tissue were observed by HE and Masson staining,and the expression of E-cadherin and ZO-1 protein in peritoneal tissue was detected by immunohistochemical(IHC).The protein expression levels of TGF-β1,BMP-7,Gremlin,E-cadherin,FN and COL-1 in peritoneal tissue were detected by WB,and the m RNA expression levels of TGF-β1,BMP-7,Gremlin,Smad1,Smad5,Smad8,Vimentin,α-SMA and IL-6 in peritoneal tissue were detected by PCR.The concentrations of TGF-β1,BMP-7and Gremlin were detected by ELISA.Results:1.The results of biochemical test showed that the levels of Scr and BUN in rats after modeling were significantly higher than those before modeling(P<0.01),indicating that the uremic model was successfully established.The results of HE staining showed that in the model group,part of the peritoneal mesothelial cells fell off,the mesothelial layer was significantly thickened,a large number of monocytes,macrophages and fibroblasts infiltrated,blood vessels proliferated,and the above changes in each drug intervention group were improved in varying degrees compared with the model group.The results of Masson staining showed that a large number of blue-stained collagen fibers deposited in the peritoneum,inflammatory cells infiltrated,neovascularization increased and the peritoneal thickness increased significantly in the model group.Compared with the model group,the degree of peritoneal collagen fiber deposition in each drug intervention group decreased in varying degrees.The results of IHC staining showed that E-Cadherin and ZO-1 proteins of tight junction proteins were widely distributed in the peritoneum of rats in the Sham group,while the distribution of E-Cadherin and ZO-1 proteins in the model group was significantly decreased.Compared with the model group,the expression of the above index proteins in each drug intervention group was significantly higher than that in the model group.2.The results of WB showed that the protein expression levels of TGF-β1,Gremlin,FN and COL-1 in the peritoneal tissue of the model group were significantly higher than those of the Sham group(P<0.01).Compared with the model group,the expression level of TGF-β 1protein in benazepril group and high dose Fushen decoction group decreased significantly(P<0.01),the levels of Gremlin,FN and protein in each treatment group decreased significantly(P<0.01),and the expression level of COL-1 protein in Fushen decoction high dose group decreased significantly(P < 0.05).Compared with the Sham group,the expression of BMP-7 and E-Cadherin protein in the peritoneal tissue of the model group decreased significantly(P<0.01),compared with the model group,the expression levels of BMP-7 and E-cadherin protein in benazepril group and high dose Fushen decoction group increased significantly(P<0.01),and the expression level of E-cadherin protein in the low dose Fushen decoction group increased significantly(P < 0.05).3.The results of PCR showed that the m RNA expression levels of TGF-β1,Gremlin,Vimentin,α-SMA and IL-6 in the peritoneal tissue of the model group were significantly higher than those of the Sham group(P<0.01).Compared with the model group,the TGF-β1m RNA expression level of benazepril group and the high dose Fushen decoction group decreased significantly(P<0.01),and the TGF-β1 m RNA expression level of the low dose Fushen decoction group decreased significantly(P<0.05).The expression levels of Gremlin,α-SMA and IL-6 m RNA in peritoneum were significantly decreased in all treatment groups(P<0.01).The expression level of Vimentin m RNA in low and high dose Fushen decoction groups was significantly decreased(P<0.01),and the expression level of Vimentin m RNA in benazepril group was significantly lower than that in benazepril group(P<0.05).Compared with the Sham group,the expression levels of BMP-7,Smad1,Smad5 and Smad8 m RNA in the peritoneal tissue of the model group were significantly decreased(P<0.01),while the expression levels of BMP-7,Smad1,Smad5 and Smad8 m RNA in the benazepril group and high dose Fushen decoction treatment group were significantly higher than those in the model group(P < 0.01).4.The results of ELISA showed that the concentrations of TGF-β1 and Gremlin in the peritoneal tissue of the model group were significantly higher than those of the Sham group(P< 0.01),and the concentrations of TGF-β1 and Gremlin of each drug intervention group were significantly lower than those of the model group(P < 0.01).Compared with the Sham group,the concentration of BMP-7 in the peritoneal tissue of the model group decreased significantly(P < 0.01),and the concentration of BMP-7 in each drug intervention group increased significantly compared with the model group(P < 0.01).Conclusion:1.Fushen decoction can reduce the degree of peritoneal injury and the deposition of peritoneal collagen fibers in PF rats,and up-regulate the expression of tight junction protein in peritoneal mesothelial cells.2.Fushen decoction can effectively intervene and reverse the EMT process of peritoneal mesothelial cells,inhibit inflammatory reaction and PF progression,and its mechanism may be related to the rebalance of TGF-β1/BMP7/Gremlin signal pathway. |
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