Gastrodin Attenuates Hypertensive Renal Fibrosis Via Suppression Of The TGF-β1/Smad2/3 Signaling Pathway

Objective:The aim of this study was to investigate the effect of gastrodin on hypertension-induced renal injury and its mechanism of action through in vivo and vivo experiments as well as network pharmacological analysis.It will further enrich the mechanism of antihypertensive effect of gastrodin and provide theoretical and experimental basis for the prevention and treatment of hypertension-induced renal fibrosis.Methods:1.In vivo experiments: Wistar Kyoto(WKY)rats and SHR rats were selected as the study subjects,and the experimental groups were: control group: Wistar Kyoto(WKY)rats(n = 5),model group: SHR group(n = 5),intervention group: SHR + Gastrodin(3.5 mg/kg/day),and rats in WKY and SHR groups were given equal amounts of double-distilled water for 10 weeks.Blood pressure and body weight of rats were measured every 2 weeks using CODA? noninvasive blood pressure monitor and electronic balance;Hematoxylin-eosin(HE)staining was used to detect the pathological changes of renal tissues;Masson’strichrome and Sirius red staining were used to detect the collagen content of renal tissues;Immunohistochemistry(IHC)staining was used to detect the protein experssions of α-smooth muscle actin(α-SMA),collagen type I(collagen I),collagen type III(collagen III),transforming growth factor-β1(transforming growth factor-β1,TGF-β1),collagen I,collagen III,p-smad2,smad2,p-smad3 and smad3 in renal tissues.2.Network pharmacology analysis: Gene Cards and Dis Ge NET databases were used to retrieve targets of gastrodin and hypertensive renal injury;Cytoscape software was used to construct protein-protein interaction(PPI)network data of "gastrodin-renal fibrosis targets";annotation,visualization and integrated discovery database(DAVID)databases were used to enrich common targets related to gastrodin and renal fibrosis into multiple functional processes GO and KEGG pathways;the most enriched fibrosis-related targets(TGF-β1 and Smad2)were selected for molecular docking with gastrodin.3.In vitro experiments: Rat kidney fibroblast cell line(NRK-49F)was stimulated with TGF-β1(5 ng/ml)and given different concentrations of gastrodin(25 μM-100 μM)intervention;CCK-8 assay was used to determine the cell viability;Immunofluorescence(IF)was used to detect the expressions of α-SMA and fibronectin protein in NRK-49 F cells;Western blot was used to assess the expressions of α-SMA,fibronectin,collagen I and TGF-β1/Smad2/3signaling pathway-related proteins in NRK-49 F cells.Results:1.In vivo experimental results: gastrodin intervention significantly suppressed the increase of systolic blood pressure(SBP),diastolic blood pressure(DBP)and mean arterial pressure(MAP)in SHR rats(P<0.05),and had no significant effect on the body weight of SHR rats(P>0.05);gastrodin intervention reduced renal injury and pathological changes in SHRs(P<0.05);gastrodin intervention reduced the accumulation of collagen in the renal tissues of SHRs by down-regulating the experssion of α-SMA,type I collagen,and type III collagen(P<0.05);gastrodin intervention significantly inhibited the activation of TGF-β1/Smad2/3signaling pathway in the renal tissues of SHR rats(P<0.05).2.Results of network pharmacological analysis: overlapping gene analysis identified 40 common genes;PPI network analysis identified TGF-β1 and Smad2 as two major candidate targets of gastrodin against hypertensive renal injury;GO and KEGG analysis showed that TGF-β1 and Smad2 proteins were heavily enriched in biological processes;Molecular docking showed high binding affinity between TGF-β1-gastrodin and Smad2-gastrodin,suggesting that gastrodin may be one of the potential inhibitory molecules for renal fibrosis.3.In vitro results: gastrodin intervention had no effect on cell viability of TGF-β1-stimulated NRK-49 F cells;gastrodin intervention significantly reversed the upregulation of α-SMA,fibronectin and collagen I protein expression in TGF-β1-stimulated NRK-49 F cells(P<0.05);gastrodin intervention significantly inhibited TGF-β1-induced upregulation of p-Smad2 and pSmad3 protein in NRK-49 F cells(P<0.05).Conclusion:Gastrodin not only inhibit the increase of blood pressure in SHR rats,but also inhibit the generation and deposition of collagen in the kidney tissue of SHR rats,thereby alleviating the renal fibrosis caused by hypertension.Gastrodin may exert its antihypertensive and renal protective effects by inhibiting the activation of TGF-β1/Smad2/3 signaling pathway.