Activation Of HIF-1? Signaling Pathway Is Associated With Cytoprotective Effects Of Salidroside Against Methylmercury-induced Toxicity In Rat Primary Astrocytes

Objective:Methylmercury(MeHg)is highly neurotoxic organic mercury compound that is particularly damaging the central nervous system(CNS),but the mechanism of neurotoxicity has not been elucidated and there are no effective treatments for MeHg poisoning.Hypoxia-inducible factor-1?(HIF-1?)is a transcription factor that plays an important role in the adaptive response,and is involved in the regulation of cell survival,proliferation,inflammation,angiogenesis,glucose metabolism,erythropoiesis,and other physiological activities.Recent studies have found that HIF-1? signaling pathway plays an important role in the occurrence and development of ischemic/hypoxia injury and various neurodegenerative diseases such as Alzheimer's disease.Therefore,HIF-1? signaling pathway has become the focus in the field of neuroprotection.Salidroside(Sal)is one of the important bioactive components extracted from Rhodiola rosea L,which has a variety of pharmacological effects including anti-inflammatory,anti-aging,and neuroprotective effects.Studies have found that the neuroprotective effect of salidroside is related to the regulation of HIF-1? signaling pathway,but its specific mechanism of action has not been fully understood.Therefore,primary rat astrocytes(AST)were used to study the role of HIF-1? signaling pathway in salidroside protecting against methylmercury-induced neurotoxicity and its mechanism.It will provide scientific basis for the treatment and prevention of methylmercury-induced neurotoxicity.Methods:1.Primary rat astrocytes were treated with different concentrations of MeHg(1,2.5,5,10 ?M,6 h),cell viability was detected by MTT assay,cytotoxicity was detected by LDH assay,apoptotic changes was detected by Hoechst 33342 staining,and the expression of HIF-1? and its pathway proteins GLUT-1,EPO,and VEGF-A was detected by Western blot.In the acute MeHg(2?4?6?8?10 mg/kg,0.5 h)exposure SD model,Western blot was used to detect the expressions of HIF-1? and its pathway proteins GLUT-1,EPO and VEGF-A in the brain tissues.2.MeHg(5 ?M,6 h)exposed cells were treated with different concentrations of Sal(5,10,20,40 ?M,24 h),cell viability was detected by MTT assay,cytotoxicity was detected by LDH assay,apoptotic changes were detected by Hoechst 33342 staining.3.The expression of cellular HIF-1? and its pathway proteins GLUT-1,EPO,VEGF-A,and heterodimeric protein HIF-1? was detected by Western blot at different concentrations of Sal(5,10,20,40 ?M,24 h).Western blot was used to detect the expression of HIF-1? and its pathway proteins GLUT-1,EPO,VEGF-A,and heterodimeric protein HIF-1? in cells in control group,MeHg(5 ?M,6 h)treated group,and Sal(20 ?M,24 h)intervention group.MTT assay was used to detect the cell viability in Sal-protected cells after HIF-1? knockdown using siRNA.4.RT-PCR was used to detect HIF-1? mRNA levels in control group,MeHg(5 ?M,6 h)exposed group,and Sal(20 ?M,24 h)intervention group.After inhibition of HIF-1? by the HIF-1? nuclear translocation inhibitor 2MeOE2,Western blot was used to detect the expression of HIF-1? protein,and MTT assay was used to detect the cell viability in Sal-protected cells.Western blot was used to detect the changes in PHD-2 protein activity in control group,MeHg(5 ?M,6 h)exposed group,and Sal(20 ?M,24 h)intervention group.5.UV-vis spectra was used to analyze the wavelength and absorbance values corresponding to the absorption peaks of Sal and MeHg complex in the range of 200-500 nm.Results:1.Compared with the control group,MeHg exposure decreased cell proliferation viability in a dose-dependent manner,increased LDH leakage rate and caused apoptosis,5 ?M MeHg treated for 6 h decreased cell proliferation viability by 44%(P<0.01),increased LDH leakage rate by 37%(P<0.01),and caused apoptosis.The results of Western blot showed that MeHg(5 ?M,6 h)significantly inhibited the expression of HIF-1? and its downstream proteins GLUT-1,EPO and VEGF-A by 51%(P<0.05),46%,31% and 41%(P<0.05),respectively.In the acute MeHg injury model,and the expression of HIF-1? and its downstream proteins GLUT-1,EPO and VEGF-A were significantly reduced at MeHg doses of 8 and 10 mg/kg(P<0.05).2.Compared with the MeHg-treated group,20 ?M Sal restored cell proliferation viability by 30%(P<0.05),LDH leakage rate was significantly reduced by 58%(P<0.05),and alleviated MeHg-induced apoptosis.3.Sal(20,40 ?M,24 h)elevated the expression of cellular HIF-1? and its pathway proteins GLUT-1,EPO,and VEGF-A(P<0.05),but different concentrations of Sal(5,10,20,40 ?M,24 h)did not significantly alter the expression of the heterodimeric protein HIF-1?.The protective effect of Sal on cell proliferation was reduced from 64% to 50%(P<0.05)after siRNA interference.4.HIF-1? mRNA levels in astrocytes were not significantly altered by MeHg exposure or Sal intervention compared with control group.HIF-1? nuclear translocation inhibitor 2MeOE2 reduced Sal protected cell viability by 22%(P<0.05).PHD-2 activity was significantly suppressed by 66%(P<0.01)in the Sal intervention group compared with the MeHg exposed group.5.The UV-vis spectra indicated that the salidroside MeHg complex did not change the chemical bond,and no new phase was produced.Conclusion:1.MeHg induced decreased cell viability,increased LDH leakage and induced apoptosis in astrocytes,and MeHg exposure decreased the expression of HIF-1? and its pathway proteins in cultured astrocytes and in the brain tissues of SD rats.2.Sal intervention mitigated MeHg-induced cell toxicity,and improved the expression of HIF-1? pathway proteins.3.Activation of the HIF-1? signaling pathway was involved in the cytoprotective effects of Sal.4.Increased HIF-1? nuclear translocation and inhibition of PHD-2-dependent HIF-1? degradation may play an important role in the accumulation of HIF-1? protein of Sal.5.The cytoprotective effect of Sal against MeHg-induced toxicity was not achieved by the coordination reaction with MeHg.