| Objective: To observe the effect of Qingshen Granules on renal fibrosis mediated by endothelial mesenchymal transition in vivo.To investigate the role of P-selectin /PSGL-1 / MAPK signaling pathway in inflammation mediated endothelial cell damage and endothelial mesenchymal transition in vitro.To study the effect of drug-contained serum of Qingshen Granules on P-selectin / PSGL-1 / MAPK signaling pathway and inflammation mediated endothelial cell injury and fibrosis.Methods: 5 / 6 nephrectomized rats were used as the model of glomerular end othelial cell injury and fibrosis.Rats were divided into Qingshen granule group,shamgroup and model group for 12 weeks' treatment.The serum levels of SCR and BUN were detected.He and Masson staining were used to evaluate the leve l of renal pathological injury.The protein levels of NLRP3 and IL-18 were dete cted by Western blot.The renal tissue was detected by immunohistochemistry forthe expression of ?-SMA protein.The expression of CD31,FSP-1 and MCP-1 w ere detected by immunofluorescence.1.LPS induced HUVEC injury was used as an inflammation mediated endothelia l injury and fibrosis model.The cells were divided into normal control group,L PS group and LPS + P-selectin inhibitor group.Cell apoptosis was detected by f low cytometry;The protein contents of P-selectin,PSGL-1,ERK1 / 2 and p-ER K1 / 2,JNK and p-JNK,p38 and p-p38,ERK5 and p-erk5 were detected by W estern blot;The m RNA levels of NLRP3 and IL-18 were detected by QRT PCR;Immunofluorescence assay was used to detect the ?-SMA,e NOS levels.2.LPS induced HUVEC injury was used as an inflammation mediated endothelia l injury and fibrosis model.The cells were divided into normal control group;L PS group;LPS + P-selectin blocking group;LPS + Qingshen granule low dose group;LPS + Qingshen granule medium dose group;LPS + Qingshen granule hi gh dose group.The protein contents of P-selectin,PSGL-1,ERK1 / 2 and p-ER K1 / 2,JNK and p-JNK,p38 and p-p38,ERK5 and p-erk5 were detected by W estern blot;QRT PCR was used to detect P-selectin,PSGL-1,e NOS,ET-1,FSP-1 ?-SMA m RNA levels.The expression of p-selectin?PSGL-1?IL-18?MCP-1?e NOS?ET-1?FSP-1??-SMA were detected by immunofluorescence.3.The active components in Qingshen granules were analyzed by ultra performa nce liquid chromatography.Results:1.1 The levels of Scr and BUN in the model group were higher than those in t he sham group(P < 0.05),and those in the Qingshen granule group were lower than those in the model group(P < 0.05).1.2 The results of he and Masson staining showed that the structure of glomeruli and renal tubules in sham group was clear,without adhesion,proliferation,fibro sis and inflammatory cell infiltration.In the model group,there were significant pathological damage,glomerular endothelial cell infiltration,glomerulosclerosis an d fibrosis,renal tubular atrophy and necrosis.In the Qingshen granule group,glo merular endothelial cell infiltration,glomerulosclerosis and fibrosis,renal tubular atrophy and necrosis were reduced compared with the model group.1.3 The relative expression of NLRP3 and IL-18 protein in model group was hi gher than that in sham group(P < 0.05),and the relative expression of NLRP3 and IL-18 protein in Qingshen granule group was lower than that in model grou p(P < 0.05).1.4 The relative expression of ?-SMA protein in model group was higher than th at in sham group(P < 0.05),and the relative expression of ?-SMA protein in Q ingshen granule group was lower than that in model group(P < 0.05).1.5 In sham group,the expression of CD31 was strongly positive(green fluoresc ence was bright),but the expression of FSP-1 and MCP-1 was weak;In the mo del group,the expression of CD31 was weak,FSP-1 and MCP-1 were strongly positive;The expression of CD31 in Qingshen granule group was higher than th at in model group,while the expression of FSP-1 and MCP-1 in Qingshen granu le group was lower than that in model group.2.1 Compared with the control group,the early and late apoptosis rates of LPS group were increased(P < 0.05);The early and late apoptotic rates of LPS + P-selectin inhibitor group were lower than those of LPS group(P < 0.05).2.2 Compared with the control group,the protein levels of P-selectin,PSGL-1,E RK1 / 2,p-ERK1 / 2,JNK and p-JNK,p38 and p-p38,ERK5 and p-erk5 were increased in LPS group(P < 0.05);Compared with LPS group,the protein lev els of P-selectin(P < 0.05),PSGL-1,ERK1 / 2,p-ERK1 / 2,JNK,p-JNK(P <0.05),p38(P < 0.05),p-p38(P < 0.05),ERK5 and p-ERK5 in LPS + P-select in inhibitor group decreased.2.3 Compared with the control group,the levels of NLRP3 and IL-18 m RNA in LPS group increased(P < 0.05);Compared with LPS group,the m RNA levels of NLRP3 and IL-18 in LPS + P-selectin inhibitor group decreased(P < 0.05).2.4 Compared with the control group,the expression of ?-SMA in LPS group w as strongly positive(red fluorescence was bright),and the expression of ?-SMA in LPS + P-selectin inhibitor group was weaker than that in LPS group.The ex pression trend of e NOS was opposite.The expression of e NOS in LPS group w as the weakest,and the expression of e NOS in LPS + P-selectin inhibitor group was between the two groups.3.1 Compared with LPS group,the protein expressions of P-selectin and PSGL-1 in all dose groups of Qingshen granules decreased in a dose-dependent manner,and the protein expressions of P-selectin and PSGL-1 in high dose group of Qi ngshen granules were the lowest(P < 0.05).Compared with LPS group,the prot ein expressions of JNK and p-JNK,p38 and p-p38 in all dose groups of Qingsh en granules were decreased,and the decrease degree of high dose group was mo re significant(P < 0.05).3.2 Compared with LPS group,the m RNA expressions of P-selectin,PSGL-1,ET-1,FSP-1 and ?-SMA in all dose groups of Qingshen granules were significantly decreased(P < 0.05),and the decrease degree of Qingshen granules high-dose group was significantly different from that of medium and low-dose group(P <0.05).Compared with LPS group,the expression of e NOS in high,medium and low dose group of Qingshen granules was significantly increased(P < 0.05),and there was significant difference among them(P < 0.05).3.3 In thecontrol group,P-selectin,PSGL-1,IL-18,MCP-1,ET-1,FSP-1 and ?-S MA were weakly expressed.In LPS group,P-selectin,PSGL-1,IL-18,MCP-1,E T-1,FSP-1 and ?-SMA were positive(red fluorescence).Compared with LPS gro up,the expression of P-selectin,PSGL-1,IL-18,MCP-1,ET-1,FSP-1 and ?-SM A in high-dose group was the weakest.In the control group,the expression of e NOS was higher than that in the LPS group.The expression of e NOS increased in all dose groups and the highest expression was found in the high dose grou p.4.Eleven active components(chlorogenic acid,berberine hydrochloride,plantain,scoparolide 6,7-dimethoxycoumarin,epiberberine,berberine,salvianolic acid B,pal matine,leonine,rhein,tanshinone IIA)in Qingshen granules were identified by u ltra performance liquid chromatography,Moreover,these eleven components could intervene and regulate inflammation,MAPK signaling pathway,cell transdifferen tiation and fibrosis.Conclusion:1.P-selectin / PSGL-1 mediates the activation of MAPK signaling pathway,initi ates inflammatory effects,leads to inflammatory injury and dysfunction of endoth elial cells,and then causes Endmt pathological changes and promotes fibrosis.2.Qingshen granule inhibited the activation of MAPK signaling pathway(p38 an d JNK pathway)mediated by P-selectin / PSGL-1,thereby reducing the inflamm atory injury of endothelial cells,reversing Endmt and delaying the process of fib rosis,and the effect was dose-dependent.3.Eleven active components which can interfere and regulate inflammation,MA PK and other signaling pathways,Endmt and fibrosis,in Qingshen granules were identified by ultra performance liquid chromatography.This supported the scienti fic nature of the research results. |
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