Objective To explore the potential mechanism of Liuwei Dihuang Decoction （LWDHD）in intervening renal fibrosis based on a network pharmacological approach and in vitro and in vivo experiments.Methods （1）To search and screen the active ingredients of LWDHD and their targets through the TCMSP platform;to screen the targets related to renal fibrosis disease through GeneCards,OMIM and TTD databases;R language VennDiagram package was used to intersect the targets of active ingredients of LWDHD and the targets related to renal fibrosis disease to obtain the common （intersecting） targets,which are LWDHD key targets for the treatment of renal fibrosis;Cytoscape 3.9.1 software was used to construct the"active ingredients-key targets"network for the treatment of renal fibrosis in LWDHD and analyze the core active ingredients;STRING platform was used to construct the common target protein-protein interaction （PPI） network;DAVID database,the R language software,and the R language software were used to analyze the core active ingredients respectively.The DAVID database and Pathview package of R language software were used for GO function and KEGG pathway enrichment analysis of the common targets.AutoDock vina software was used to perform molecular docking between core active ingredients and core targets.（2）In vivo experiments:24 male SD rats were randomly divided into 4 groups:shamoperation group,model group,LWDHD group (6.75 g·kg -1 )and Enalapril group (10 mg·kg -1 ),6 rats in each group.A rat model of renal fibrosis was established using unilateral ureteral ligation （UUO）.The rats were administered by gavage (10 mL·kg -1 )once daily for 7 consecutive days.The renal pathological changes and collagen deposition were observed by HE and MASSON staining.（3）In vitro experiments:HK-2 cells cultured in vitro were divided into normal control group （10%blank serum）,model group (10%blank serum+200μmol·L -1 CoCl 2 )and LWDHD group (10%drug-containing serum+200μmol·L -1 CoCl 2 );200μmol·L -1 CoCl 2 was used to simulate the intrarenal hypoxic environment,while the cells were collected after 24 hours of incubation with either blank or drugcontaining serum;the HIF-1α protein expression level of HK-2 cells was detected by Western Blot.Results （1）A total of 40 LWDHD active ingredients,194 active ingredient action targets,5 135 renal fibrosis disease-related targets and 159 common （intersecting）targets,i.e.key targets of LWDHD for the treatment of renal fibrosis were obtained.The core active ingredients screened were quercetin,stigmasterol,kaempferol,β-sitosterol and diosgenin,etc.The core targets were heat shock protein 90α family class A member 1 （HSP90AA1）,c-Jun aminoterminal kinase （JUN）,protein kinase B （AKT1）and hypoxia-inducible factor-1α （HIF-1α）,etc.GO function was mainly related to cytokine mediated signaling pathways,positive regulation of gene expression,negative regulation of apoptosis,hypoxia response and other biological processes;KEGG key pathways include atherosclerosis,cancer pathway,viral infection,advanced glycosylation end product-receptor for glycosylation end product （AGE-RAGE） pathway,tumor necrosis factor （TNF） signaling pathway,interleukin 17 （IL-17）signaling pathway,HIF-1 signaling pathway,etc.Quercetin binds moderately to HSP90AA1 and HIF-1α,stigmasterol binds moderately to HSP90AA1,JUNE,AKT1 and HIF-1α,and diosgenin element has strong binding to HIF-1α.（2）In vivo experiments showed that compared with the sham-operation group,the model group rats showed interstitial inflammatory cell infiltration,vacuolization,tubular atrophy and compensatory dilation of some tubules;renal tissue fibrosis was evident,with a large number of collagen fibres stained blue and a significantly higher collagen volume fraction seen （P＜0.01）.Compared with the model group,the LWDHD and Enalapril groups showed less kidney tissue lesions,less inflammatory cells and less structural damage to the renal tubules;the blue collagen fibers in the kidney tissue were significantly reduced,the degree of fibrosis was significantly reduced and the collagen volume fraction was significantly decreased （P＜0.01）.（3） In vitro experiments showed that the HIF-1α protein level in the model group was significantly increased compared with the normal group （P＜0.01）;compared with the model group,the HIF-1α protein expression level in the LWDHD group was significantly decreased （P＜0.05）.ConclusionThe therapeutic effect of LWDHD on renal fibrosis may be achieved through multiple active ingredients such as quercetin,stigmasterol and diosgenin,acting on multiple targets such as HSP90AA1,JUNE,AKT1 and HIF-1α,through multiple signaling pathways such as AGE-RAGE signaling pathway,TNF signaling pathway,IL-17 signaling pathway and HIF-1 signaling pathway.

Objective To observe the effects and mechanism of Liuwei Dihuang Decoction（LWDHD） regulating epithelial mesenchymal transition（EMT） of HK-2 cells induced by CoCl2through miR-210/HIF-1α signaling pathway. Methods HK-2 cells were cultured in vitro and divided into normal group（N group）, model group（M group）, LWDHD medicated serum group（LW group）,blank serum+over-espressed miR-210 group（LV-miR-210 group）, blank serum+silent miR-210 group（LV-anti-miR-210 group）,blank serum+miR-210 negative group（LV-miR-210 NC group）, LWDHD medicated serum+over-expressed miR-210 group（LW+LVmiR-210 group）, LWDHD medicated serum+silent miR-210 group（LW+LV-anti-miR-210 group） and LWDHD medicated serum+miR-210 negative group（LW+LV-miR-210 NC group）. Except for N group, the other groups were treated with CoCl2. CCK-8 was used to detect the effect of different concentrations of LWDHD and CoCl2on the activity of HK-2 cells after 24 hours, and the best intervention concentration was selected. Cell immunofluorescence and Western blot were used to determine hypoxia-inducible factor-1α（HIF-1）, β-catenin, Vimentin, and E-cadherin protein expression levels. miR-210, HIF-1α, β-catenin and Vimentin mRNA expression levels were measured by qPCR. By lentivirus transfection of HK-2 cells, miR-210 was overexpressed and inhibited. Then Co Cl2was added to the medium. The miR-210, HIF-1α, β-catenin, Vimentin protein and mRNA expression of the regulation effect of LWDHD were observed. Results The CCK-8 showed that the optimal intervention concentrations of LWDHD and CoCl2were 10% and 200 μmol/L, respectively. Compared with N group, the morphology of cells in group M changed from the shape of paving stone to the shape of long spindle. HIF-1α, β-catenin, Vimentin protein and mRNA expression increased（P＜0.05,P＜0.01） while miR-210 mRNA expression increased（P＜0.01） and E-cadherin protein expression decreased（P＜0.01）. Compared with M group, LW group can significantly reduce the expression of HIF-1α, β-catenin, Vimentin protein and mRNA（P＜0.05, P＜0.01）, downregulate the expression of miR-210 mRNA（P＜0.05） and up-regulate the expression of E-cadherin protein（P＜0.01）. Compared with the LV-miR-210 NC group, LV-anti-miR-210 group showed lower HIF-1α, β-catenin, Vimentin protein and m RNA（P＜0.05, P＜0.01）, and LV-miR-210 group showed higher HIF-α, β-catenin, Vimentin protein and mRNA（P ＜0.05, P ＜0.01）. After LWDHD administration to each group of lentivirus transfection, HIF-1α, β-catenin, Vimentin protein and mRNA expression decreased（P＜0.05, P＜0.01）. Conclusion The anti-fibrosis mechanism of LWDHD is related to its down-regulation of miR-210/HIF-1α signaling pathway, which is related to the inhibition of renal tubular EMT.

OBJECTIVES:The nuclear factor-κB (NF-κB) signaling pathway plays an important role in regulating tubular epithelial-mesenchymal transition (EMT), an indispensable cellular programme for driving organ fibrosis and tumor progression. Liuwei Dihuang Decoction (LWD) is an effective Chinese formula for treating chronic renal failure.METHODS:First, by using morphological examination, immunofluorescence staining assay, RTqPCR, and Western blot analysis, in vitro experiments were designed to analyze NF-κB and EMT markers (including Snail, α-SMA, and E-cadherin) in transforming growth factor-β1 (TGF-β1) induced renal tubular epithelial cells (HK-2) and to detect the expression levels of LWD-CS cotreatment. Then, the recombinant lentiviral vector was overexpressed and knocked down by NF- κB and transfected into HK-2 cells. Cells were treated with TGF-β1 (10 ng/ml) with blank serum or LWD-containing serum, respectively, and the expression of these molecules in the NF-κB/Snail signaling pathway was further evaluated.RESULTS:Our results confirmed that TGF-β1 could induce EMT, nuclear translocation of NF-κB p65, and activate the NF-κB/Snail signaling pathway in HK-2 cells. Furthermore, NF-κB knocked-down dramatically increases the TGF-β1-induced mRNA and protein expression level of E-cadherin and reduces the level of Snail and α-SMA; this is reversed by NF-κB overexpression. LWD can decrease the EMT levels through the NF-κB/Snail signaling activation in TGF-β1-induced EMT of HK-2 cells.CONCLUSION:The present study provides evidence suggesting a novel mechanism that LWD exerts anti-fibrosis effects through inhibiting activation of the NF-κB/Snail signaling pathway and consequently downregulating the TGF-β1-induced EMT in renal tubular epithelial cells.

目的 观察六味地黄汤含药血清对CoCl2缺氧后HK-2细胞中的上皮-间充质转化(epithelial-mesenchymal transition,EMT)标志物E-钙黏蛋白(E-cadherin)和α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的影响.方法 体外培养HK-2细胞,分为3组:正常组(10％空白血清)、模型组(10％空白血清+200μmol·L-1 CoCl2)、含药血清组(10％含药血清+200μmol·L-1 CoCl2).CCK-8法检测不同浓度空白血清、含药血清(2.5％、5％、10％、20％、40％)在24、36 h对HK-2细胞存活率的影响;以及不同浓度CoCl2(150、200、300、600μmol·L-1)在24 h的最佳干预浓度.qRT-PCR法和Western blot法分别检测各组E-cadherin、α-SMA的mRNA和蛋白表达水平;免疫荧光观察各组E-cadherin、α-SMA蛋白表达情况.结果 CCK-8法确定含药血清和空白血清的最佳干预浓度和干预时间分别为10％和24 h;CoCl2的最佳干预浓度为200μmol·L-1.与正常组比较,模型组E-cadherin的mRNA和蛋白表达水平均明显降低(P<0.01)、α-SMA明显升高(P<0.05);与模型组比较,含药血清组E-cadherin的mRNA和蛋白表达水平均明显升高(P<0.01)、α-SMA明显降低(P<0.01).结论 缺氧能诱导HK-2细胞发生EMT,六味地黄汤含药血清能够降低α-SMA的表达,升高E-cadherin的表达,从而缓解肾小管EMT.