Nrf2 Knockout Attenuates The Effects Of Astragaloside Ⅳ Through PSmad3C/3L Pathways Against Kidney Fibrosis

BackgroundsThe kidneys are important for maintaining homeostasis and excreting metabolites in the body.However,nephrotoxicity of drugs and other organ pathologies are highly likely to cause kidney injury.For example,in the treatment and research of liver cancer,it has been found that along with the formation of liver cancer,the kidneys can also be damaged.Liver cancer is a serious threat to human health,and in its treatment and research have found that along with the formation of liver cancer,the kidneys can also be damaged.Fibrotic kidney injury from hepatocarcinogenesis seriously impacts treatment effect.Nrf2/HO-1 is a key antioxidant stress pathway and help treatment of kidney injury.Nuclear erythroid 2-related factor 2（Nrf2）is a key anti-oxidative stress transcription factor that plays a role in the treatment of kidney injury by antioxidant damage,reducing cytotoxicity and thus protecting the kidney.Smad3 phosphorylation is implicated in hepatocarcinogenesis.Previous studies by our group have shown that different phosphorylated forms of Smad3 and activation of the Nrf2/HO-1 pathway regulate the progression of hepatocellular carcinoma in different directions.Astragaloside Ⅳ（AS-Ⅳ）,an extract of Astragalus membranaceus,has several pharmacological activities,which is useful in the treatment of edema and fibrosis.Therefore,in the present study,we designed experiments in which HK-2 cells were stimulated with TGF-β1 and intervened with AS-Ⅳ.Further study of AS-Ⅳ treatment of renal injury in Nrf2 knockout mice during hepatocarcinogenesis and its mechanism of action.Investigation of the therapeutic effect of AS-Ⅳ on kidney damage by hepatocarcinogenesis.The focus is on whether AS-Ⅳ treatment with Smad3 phosphorylation and the Nrf2/HO-1 signal pathway are involved and whether there is an effect of Nrf2 deletion on Smad3 phosphorylation.Objectives1.To investigate whether AS-Ⅳ can reduce oxidative injury and fibrotic stimulation of HK-2 cells by TGF-β1 through regulation of Nrf2/HO-1 and pSmad3C/3L signal pathways.2.To observe whether kidney damage is exacerbated in mice when Nrf2 is knocked out,and the effect of AS-Ⅳ treatment.3.To clarify the effect of Nrf2 knockdown on the pSmad3C/3L signal pathway and to explore the effect of Nrf2 loss on the regulation of the pSmad3C/3L signal pathway by AS-Ⅳ.Methods1.AS-Ⅳ on HK-2 cells reduces oxidative injury and fibrotic stimulation of TGF-β1 through modulation of Nrf2/HO-1 and pSmad3C/3L signal pathways.Grouped HK-2 cells as follows:control group,TGF-β1 group（10 ng/ml）,TGF-β1（10 ng/ml）+AS-Ⅳ（10μM）group,TGF-β1（10 ng/ml）+AS-Ⅳ（20μM）group,TGF-β1（10 ng/ml）+AS-Ⅳ（40μM）group.TGF-β1 and AS-Ⅳ were added to the cells for24h and the remaining groups were treated with the corresponding dose of vehicle.Used DCFH-DA to detect ROS level in HK-2 cells;cellular immunofluorescence to examine the expression and distribution of Nrf2,p Nrf2,HO-1,pSmad3C and pSamd3L proteins in HK-2 cells;and Western blot assay was performed to detect the expression of related proteins in fibrosis marker（α-SMA）,Nrf2/HO-1 and pSmad3C/3L signal pathways in HK-2 cells.2.Establishment of an Nrf2 knockout model of renal injury caused by hepatocellular carcinoma formation and investigation of the effect on renal injury treated by AS-Ⅳ though pSmad3C/3L signal pathwayNrf2 knockout mice were reared and expanded in SPF-class animal center,and the mice were genetically identified.Select 18 each of 6th generation homozygous Nrf2knockout C57BL/6 mice（HO）and Homologous wild type（WT）and randomly group them as follows:WT-Control,WT-Model,WT-Model-AS-Ⅳ,HO-Control,HO-Model and HO-Model-AS-Ⅳ groups.Combined mold making with DEN/CCl₄/C₂H₅OH,DEN（100 mg/Kg,1-2 weeks,1times/week,ip）,20%CCl₄（0.05 ml/10g,3-7 weeks,2 times/week,ig）;20%CCl₄（0.06 ml/10g,8-18 weeks,2 times/week,ig）;10%alcohol instead of drinking water（3-18 weeks,change every other day）.The treatment group was given AS-Ⅳ（40 mg/Kg,ig）in every day and the rest of the group was given the corresponding dose of vehicle.At 12 and 20 weeks,we performed HE/Masson staining after collection;biochemical indicators were used to test BUN,Serum creatinine（Scr）,MDA and SOD;p Nrf2,pSmad3C and pSmad3L protein expression and distribution in kidney tissues were measured by animal immunofluorescence.Western blot assay was performed to detect the expression of related proteins inα-SMA,TGF-β1,Nrf2/HO-1and pSmad3C/3L signal pathways in kidney tissues.Results1.AS-Ⅳ inhibits ROS levels andα-SMA expression in Hk-2 cells stimulated by TGF-β1The results of the reactive oxygen species assay andα-SMA protein expression levels showed that AS-Ⅳ further inhibited TGF-β1 stimulated oxidative and fibrotic damage in HK-2 cells with increasing doses.2.AS-Ⅳ reduces TGF-β1 stimulation of HK-2 cells by activating the Nrf2/HO-1signal pathwayWestern blot protein assay results showed that Nrf2,p Nrf2,HO-1 protein expression increased with increasing concentrations after AS-Ⅳ dosing.The results of immunofluorescence detection of Nrf2 and p Nrf2 protein expression further suggest that AS-Ⅳ can reduce the stimulation of HK-2 cells by TGF-β1 by promoting the expression of the Nrf2/HO-1 signal pathway.3.AS-Ⅳ reduces TGF-β1 stimulation of HK-2 cells by promoting activation of the pSmad3C/p21 signal pathway and inhibiting conversion of the pSmad3L/PAI-1signal pathwayWestern blot examined protein expression associated with the pSmad3C/3L signal pathway.The results showed that all proteins increased after TGF-β1 stimulation,but pSmad3C and p21 proteins diverged from pSmad3L and PAI-1 proteins after AS-Ⅳ administration.Expression of pSmad3C and p21 proteins increased further with increasing AS-Ⅳ concentrations,whereas pSmad3L and PAI-1 proteins decreased with increasing AS-Ⅳ concentrations.Immunofluorescence detection of pSmad3C and pSmad3L protein expression trends were consistent with the results of Western blot.4.Nrf2 knockout exacerbates renal injury and inhibiting the effect of AS-Ⅳ treatmentFirstly,the knockdown validation Western blot was performed at 20 weeks mice to detect the protein expression associated with the Nrf2 knockdown.And the results showed that in the HO mice group Nrf2,p Nrf2 and HO-1 proteins were not expressed and the gene knockout was successful.In addition,Nrf2,p Nrf2 and HO-1 were found to be increased after model in the WT group,and the increase was more pronounced after AS-Ⅳ administration,suggesting that AS-Ⅳ promotes the activation of Nrf2/HO-1signal pathway in WT mice.At 12 weeks and 20 weeks pathological analysis in kidneys,we was found that the kidneys were oedema at 12 weeks and oedema was more severe in the HO group,but oedema was reduced after AS-Ⅳ administration.At 20 weeks,the tubules in the kidneys showed glassy degeneration,and tubular damage was further aggravated especially in the model group of HO mice,and HO mice did not recover as much as WT mice after AS-Ⅳ administration.The Masson results showed an increase in blue staining of the kidney tissue and interstitial fibrosis after modelling,especially after HO modelling.After AS-Ⅳ administration the blue staining decreased,but the HO mice did not recover as much as the WT mice.The 20 weeks mice western blot assay showed an increase in bothα-SMA and TGF-β1 after modeling,especially in HO mice,and a decrease after AS-Ⅳ administration,especially in WT mice.For BUN and Scr results showed elevations after both 12 and 20 weeks of modeling,especially in the 20 weeks HO group,which could be reduced after AS-Ⅳ administration,but less after 20 weeks of HO administration.MDA and SOD trends were reversed,with MDA levels increasing after modeling and decreasing after administration,and SOD levels decreasing after modeling and increasing after administration.The mice in the 20-week HO group had the highest increase in MDA and the lowest decrease in SOD after modeling,and also did not recover as well as the other groups after dosing.5.Nrf2 knockdown inhibits activation of pSmad3C/p21 signaling channels and reduces the ability of AS-Ⅳ to upregulate pSmad3C/p21 signaling channels during the treatment of renal injuryWestern blot detection of pSmad3C and p21 protein levels on renal tissue at 20weeks revealed that both pSmad3C and p21 protein levels were increased to varying degrees after modeling,and the increase was more pronounced after AS-Ⅳ administration.However,none of the increased levels in the HO group were as high as the corresponding WT group.The expression trend of pSmad3C by immunofluorescence was consistent with the Western blot results.6.Nrf2 knockdown promotes activation of the pSmad3L/PAI-1 signal pathway and reduces the ability of AS-Ⅳ to downregulate the pSmad3L/PAI-1 signal pathway during treatment of renal injuryWestern blot detection of pSmad3L and PAI-1 protein levels on renal tissue at 20weeks revealed that both pSmad3L and PAI-1 protein levels were increased to varying degrees after modeling and decreased to varying degrees after AS-Ⅳ administration.The results showed that pSmad3L and PAI-1 expression were both higher in the HO modelling than in the WT model group,and were not reduced as much as in the WT administration group after AS-Ⅳ administration.The expression trend of pSmad3L detected by immunofluorescence was consistent with the Western blot results.Conclusions1.AS-Ⅳ can reduce oxidative injury and fibrotic stimulation of HK-2 cells by TGF-β1through regulation of Nrf2/HO-1 and pSmad3C/3L signal pathways.2.AS-Ⅳ regulates Nrf2/HO-1 and pSmad3C/3L signal pathways to reduce kidney fibrosis caused by hepatocarcinogenesis3.Nrf2 knockdown affects the expression of pSmad3C/3L signal pathway and affects the AS-Ⅳ regulation of pSmad3C/3L signal pathway,inhibiting the therapeutic effect of AS-Ⅳ.