| OBJECTIVE: To study the intervention of tanshinone IIA combined with DAPT on the expression of α-smooth muscle actin(α-SMA)and transforming growth factor-β(TGF-β)and Notch1/Jagged1 signaling pathway in UUO rat kidney and HSA-induced human renal tubular epithelial cells(HK-2).It is confirmed the therapeutic mechanism of tanshinone IIA combined with DAPT in preventing and treating renal fibrosis,and provided theoretical basis and experimental basis for the distribution of Chinese combined with Western medicines.Methods: 1.40 male Sprague-Dawley rats were randomly divided into 5 groups: normal group,model group,tanshinone IIA group,γ-secretase inhibitor(DAPT)group,tanshinone IIA and DAPT group(referred to as tanshinone IIA+DAPT group).The corresponding drug intervention was carried out for 14 days.The renal function indexes of rats: urine microalbumin(UMA),N-acetyl-β-D-glucosaminidase(NAG),and renal tissue histopathology were observed.Immunohistochemistry(IHC)detect the changes of α-smooth muscle actin and type I collagen(ColI).Real-time quantitative PCR(RT-PCR)was used to detect the expression of Notch1 mRNA and Jagged1 mRNA.Western Blot(WB)was used to detect Notch1,Jagged1,α-SMA and TGF-β,CTGF protein expression.2.HK-2 cell subculture after synchronization was also divided into the above five groups,except for the normal group were given excessive HSA modeling,and three drug intervention groups were given corresponding drug intervention for 48 h.The expression of Notch1,Jagged1,α-SMA,TGF-β and CTGF protein was detected by immunofluorescence.WB was used to detect the protein expression of Notch1,Jagged1,α-SMA,TGF-β,CTGF.Results: The results of renal tubular function test in each group: Compared with the normal group,the urine NAG and UMA levels in the model group were significantly increased(P<0.05);compared with the model group,the UMA in administration groups were significantly reduced(P<0.05).There was no significant difference in urinary NAG between administration groups(P>0.05).Renal pathological results of rats in each group: The renal tissue of the model group showed tubular atrophy,and the tubulointerstitial was significantly widened.Masson staining showed a large amount of collagen fibers diffusely distributed.The pathology of administration groups were improved compared with the model group,and the HE and Masson scores were significantly lower(P<0.05).The effect of the tanshinone IIA + DAPT group was obvious.3.Immunohistochemical detection of renal tissue results: Compared with the normal group,the mean optical density values of α-SMA and ColI in the renal tissue of the model group were significantly increased(P<0.05);compared with the model group,α-SMA,The average optical density of ColI decreased significantly(P<0.05).The comparison between administration groups,the effect of tanshinone IIA + DAPT group was significant(P<0.05).The results of RT-PCR detection of renal tissue: Compared with the normal group,the relative expression of Notch1 and Jagged1 mRNA in the model group was significantly increased(P<0.05).Compared with the model group,the expression of Notch1 and Jagged1 mRNA in each administration group was reduced(P<0.05).The relative expression of Jagged1 mRNA in tanshinone IIA+DAPT group was lower than that in tanshinone IIA group(P<0.05),and there was no significant difference with DAPT group(P>0.05).The relative expression of Notch1 mRNA in the kidney tissue of rats in tanshinone IIA+DAPT group was significantly lower than that of the tanshinone IIA and DAPT groups(P<0.05).Western blot analysis showed that compared with the normal group,the expression of Notch1,Jagged1,α-SMA,TGF-β and CTGF protein in the model group was significantly increased(P<0.05);compared with the model group The relative expression of Notch1,Jagged1,α-SMA,TGF-β and CTGF protein in renal tissue of each administration group was significantly decreased(P<0.05).Compared with the tanshinone IIA+DAPT group,the expression level of Notch1,α-SMA and TGF-β was significantly lower than the tanshinone IIA and DAPT groups(P<0.05).In vitro results 6.Immunofluorescence detection of HK-2 cells: The fluorescence signal of HK-2 nuclei was blue,while the fluorescent signals positively expressed by Notch1,Jagged1,α-SMA,TGF-β and CTGF were red.Notch1,Jagged1,α-SMA and CTGF showed different degrees of red fluorescence in the cytoplasmic region of each group.TGF-β showed different degrees of red fluorescence in the nucleus of each group,indicating the expression of Notch1,Jagged1,α-SMA and CTGF.In the cytoplasm of HK-2 cells,TGF-β is mainly expressed in the nucleus of HK-2 cells.7.Western blot analysis of HK-2 cells: Compared with the normal group,the relative expression of Notch1,Jagged1,α-SMA,TGF-β and CTGF protein in the model group was significantly increased(P<0.05);compared with the model group,the relative expression of Notch1,Jagged1,α-SMA,TGF-β and CTGF protein in the administration groups were significantly decreased(P<0.05).Tthe relative expression of Notch1,α-SMA,TGF-β and CTGF protein in the tanshinone IIA+DAPT groupt were significantly lower than tanshinone IIA and DAPT groups(P<0.05).There was no significant difference in the relative expression of Jagged1 between the administration groups(P>0.05).Conclusion: Tanshinone IIA,DAPT and their combination can alleviate renal tissue damage in UUO rats,protect renal tubular function and delay renal fibrosis.This mechanism may reduce the expression of α-SMA and ColI in kidney tissue,reduce the synthesis of collagen,inhibit the expression of TGF-β and CTGF in related fibrotic molecules,and effectively interfere with the multi-links of Notch1 and Jagged1 signaling pathways in renal tissues,thereby exerting its multi-target effect and delaying the progression of renal fibrosis in UUO rats.In vitro assays for albumin-induced HK-2 cells further confirmed that tanshinone IIA,DAPT and their combination may delay renal fibrosis,which may released with the reduced expression of related pro-fibrotic factors,and interfere with the multi-link and multi-target effects of Notch1 and Jagged1 signaling pathways. |
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