| Objective:Renal fibrosis is a pathological manifestation of the end stage of chronic kidney disease(CKD),characterized by abnormal deposition of extracellular matrix,prevention of renal fibrosis is one of the effective strategies to stop the progression of CKD.The mechanism of renal fibrosis is complex,and the effect of single-target inhibition is not ideal,so the holistic treatment concept of multi-target intervention in TCM is gaining more and more attention in the prevention and treatment of renal fibrosis.At present,Shenkang injection(SKI)is a commonly used and effective Chinese medicine injection for the treatment of CKD,but its specific molecular mechanism of action against fibrosis is not yet clear.In this study,we used network pharmacology to analyze the drug pathway of SKI to interfere with renal fibrosis,combined with animal and cellular experiments to verify its mechanism of action and provide a scientific basis for SKI to treat CKD.Methods:1.The TCMSP,Gene Cards,OMIM,and Dis Ge NET databases were used to obtain the active ingredient of SKI and renal fibrosis-related genes,analyze the active ingredient-drug disease interaction targets of SKI and construct a protein interaction network based on the obtained key targets,GO and KEGG analyses were also performed.2.A rat renal fibrosis model was established using unilateral ureteral obstruction(UUO)and randomly divided into the normal group(without any treatment),model group(left ureter ligation),sham group(same surgery as the model group but without ureter ligation),benazepril group(0.9 mg/kg/d),and high(5 g/kg/d),medium(2.5 g/kg/d),low(1.25 g/kg/d)dose SKI group(n=6 in each group).The intervention was administered three days after successful molding for three consecutive weeks.The rats were observed for general condition,weighed for body and kidney wet weight,and kidney function was also measured.Hematoxylin-eosin staining(HE)and Masson staining were performed on kidney tissues.The expression of proteins and genes related to ECM,EMT,and Wnt/β-catenin signaling pathway in kidney tissues were detected by immunohistochemistry,immunofluorescence,Western blot,and RT-q PCR.3.HK-2 cells were divided into the blank group(complete medium),model group(TGF-β1 at a final concentration of 10 ng/m L),SKI group,and ICG-001 group.The effect of SKI on the expression related to the cellular Wnt/β-catenin signaling pathway was detected by immunofluorescence staining,Western blot,and RT-q PCR after 24 h of intervention.HK-2 cells were divided into the blank group,model group,SKI group,and ICG-001 group.The effects of SKI on the EMT of HK-2 cells were evaluated by Phalloidin staining,Scratch test,immunofluorescence staining,Western blot,and RTq PCR.Results:1.The network pharmacology study showed that there were 135 main active ingredients of SKI,corresponding to 307 targets.Among them,there were 119 key targets overlapping with those related to renal fibrosis.Quercetin,luteolin,apigenin,kaempferol,daidzein,aloe-emodin,formononetin,and isorhamnetin may be the main components of the anti-fibrosis of SKI.The results of enrichment analysis suggest that SKI may act through several biological processes such as regulation of cell migration,cell proliferation and apoptosis,cell adhesion,and oxygen level regulation and it may act on the HIF-1,Fox O,JAK-STAT,T cell receptor,TGF-beta,Wnt,Hippo,and other signaling pathways to exert therapeutic effects.2.Animal experiments: After three weeks,the general condition of the rats in the high-dose SKI group(SKI-H),medium-dose SKI group(SKI-M),low-dose SKI group(SKI-L),and benazepril group improved,with the increased in body weight(P < 0.01)and decreased in kidney wet weight(P < 0.05)and a significant decreased in serum creatinine and urea nitrogen(P < 0.01)compared to the model group.HE staining: In the model group glomerular hyaline degeneration,tubular atrophy,some tubules and collecting ducts dilatation,vacuolization of tubular epithelial cells,the proliferation of interstitial fibroblasts,and extensive inflammatory cell infiltration.The SKI-H,SKI-M,SKI-L,and benazepril groups showed reduced histological changes as described above compared to the model group.The number of glomeruli was significantly reduced in the model group compared to the normal and sham groups(34.17 ± 6.21 versus,88.33± 8.50,88.00 ± 7.32,P < 0.01).In the SKI-H,SKI-M,SKI-L,and benazepril groups the number of glomeruli was increased compared to the model group(51.50 ± 4.59,46.00 ± 3.23,43.33 ± 5.16,49.83 ± 5.91,versus 34.17 ± 6.21,P < 0.01).Masson staining: Blue collagen material deposition was observed in the interstitium of the rats’ kidneys in the model group.The deposition was reduced in the SKI-H,SKI-M,SKI-L,and benazepril groups compared to the model group.The collagen volume fraction(CVF)of renal tissue was significantly increased in the model group compared to the normal group sham groups(50.45% ± 5.39 versus,7.13% ± 3.60,7.64% ± 2.50,P< 0.01).And CVF was significantly decreased in the SKI-H,SKI-M,SKI-L groups,and benazepril group compared to the model group(27.57% ± 5.19,28.66% ± 4.44,35.25% ± 2.97,27.89% ± 4.29,versus 50.45% ± 5.39,P < 0.01).Extracellular matrix expression: Immunohistochemistry showed that significant Col Ⅰ and FN positive particle deposition was observed in the kidney tissue of the model group.The SKI-H,SKI-M,SKI-L,and benazepril groups showed reduced positive deposition compared with the model group.Col Ⅰ and FN expression were significantly increased in the model group compared with the normal and sham groups(P < 0.01),while Col Ⅰ and FN expression were decreased in the SKI-H,SKI-M,SKI-L,and benazepril groups compared with the model group(P < 0.05).EMT-related expression:Immunohistochemistry showed that α-SMA-positive granule deposition was observed in the kidney tissue of the model group,but E-cadherin-positive granule deposition was rare.The SKI-H,SKI-M,SKI-L,and benazepril groups showed α-SMA-positive deposition was decreased and E-cadherin-positive deposition was increased compared with the model group.α-SMA expression was significantly increased(P < 0.01),and E-cadherin expression was significantly decreased(P < 0.01)in the model group compared to the normal and sham groups.The SKI-H,SKI-M,SKI-L,and benazepril groups showed decreased α-SMA expression(P < 0.05)and increased E-cadherin expression(P < 0.05)compared to the model group.Expression of Wnt/β-catenin signaling pathway: Immunofluorescence showed that multiple cells of UUO rats kidney tissue observed β-catenin expressed.The active β-catenin positive fluorescence signal was stronger in the model group than in the sham group.The active β-catenin fluorescence signal was diminished in the SKI-H,SKI-M,SKI-L,and benazepril groups compared with the model group.Immunohistochemistry showed increased deposition of β-catenin-positive particles in the kidney tissue in the model group compared to the normal and sham groups.The SKI-H,SKI-M,SKI-L,and benazepril groups showed decreased β-catenin deposition compared to the model group.Western blot protein semi-quantitative and RT-q PCR m RNA quantification showed the expression of pathway-related proteins(Wnt1,β-catenin,active β-catenin,Dvl1,TCF4,PAI-1,and Snail1)and genes(β-catenin,Wnt1,PAI-1,Snail1,and TCF4)were significantly increased in the model group(P < 0.01).The levels of pathway-related proteins and gene expression were decreased in the SKI-H,SKI-M,SKI-L group,and benazepril group compared with the model group(P < 0.05).3.Cellular experiments: Immunofluorescence staining showed that β-catenin and active β-catenin fluorescence signals were enhanced in the cytoplasm of the model group compared to the blank group.Scattered active β-catenin and Snail1 fluorescence signals were observed in the nucleus.In the SKI and ICG-001 groups,the signals of β-catenin,active β-catenin,and Snail1 were weakened compared with the model group.The expression of pathway-related proteins(β-catenin,active β-catenin,PAI-1,Snail1,TCF4,Dvl1,and Wnt1)and genes(β-catenin,PAI-1,Snail1,TCF4,and Wnt1)was significantly increased in the model group compared to the blank group by Western blot protein semi-quantitative and RT-q PCR m RNA quantification(P < 0.01).The expression of pathway-related proteins(β-catenin,active β-catenin,PAI-1,Snail1,TCF4,Dvl1)and genes(β-catenin,PAI-1,Snail1,TCF4)was decreased in the SKI and ICG-001 groups compared with the model group(P < 0.05).Wnt1 expression was significantly decreased in the SKI group compared with the model and ICG-001 groups(P < 0.05).The Phalloidin staining showed that HK-2 cells in the blank group were ovoid,and the model group had a shuttle-shaped cell morphology.Most of the cells in the SKI group had reduced shuttle-shaped changes compared with the model group.A few of cells in the ICG-001 group recovered their morphology,and the rest of the cells did not change significantly.The cell Scratch test showed that the scratch model was successfully constructed in all groups after 0 h,and there was no significant difference between the groups.After 24 h the scratch ratio was measured,and the scratch ratio in the model group increased significantly compared with that in the blank group(0.8756± 0.0500 compared to 0.2858 ± 0.1692,P < 0.01).The scratch ratio in the SKI group and ICG-001 group decreased compared with that in the model group(0.2888 ± 0.1205,0.6971 ± 0.1929,compared to 0.8756 ± 0.0500,P < 0.05),the scratch ratio in the SKI group was significantly lower than that in the ICG-001 group(P < 0.01).Immunofluorescence showed that the Col Ⅰ and FN fluorescence signals were enhanced in the model group compared with the blank group.In the SKI and ICG-001 groups,Col Ⅰ and FN fluorescence signals were weakened compared with the model group.In the model group,the cytoplasm α-SMA fluorescence signal was enhanced compared with the blank group,while the cell membrane E-cadherin fluorescence signal was weakened.In the SKI and ICG-001 groups,the cytoplasm α-SMA fluorescence signal was weakened compared with the model group,while the E-cadherin fluorescence signal was enhanced.Western blot protein semi-quantitative,RT-q PCR m RNA quantitation showed that the expression of Col Ⅰ,FN,and α-SMA were significantly increased(P < 0.01),and E-cadherin expression was significantly decreased(P < 0.01)in the model group compared with the blank group.Expression of Col Ⅰ,FN,and α-SMA decreased significantly in the SKI and ICG-001 groups,compared with the model group(P < 0.01),while the expression of E-cadherin increased(P < 0.05).Conclusion:1.Network pharmacological analysis suggests that SKI has various active ingredients that can improve renal fibrosis through diverse targets and multi-signaling pathways,Wnt signaling pathway is one of the important signaling pathways predicted to be obtained.2.SKI could improve the general condition of UUO rats,protect renal function,reduce renal pathological damage,and improve interstitial fibrosis.The Wnt/β-catenin signaling pathway was activated in UUO rats,and SKI down-regulated the expression levels of the main factors involved in this signaling pathway.3.The Wnt/β-catenin signaling pathway was activated in HK-2 cells after TGF-β1stimulation.The cell morphology was changed,and migration was significant.SKI may inhibit EMT by regulating the Wnt/β-catenin signaling pathway to exert an anti-fibrosis effect. |
PDF Full Text Request