Effect And Mechanism Of Resveratrol On Shh Signaling Pathway In NIH3T3 Cells

Objective:To investigate the effect and mechanism of resveratrol on Shh signaling pathway in NIH3T3 cells in vitro.Methods:NIH3T3 cells were used as model cells and treated with drugs.(1)To investigate the effect of resveratrol on viability of NIH3T3 cells after starvation for 24h,three groups were studied:control group(Ctrl),resveratrol group(0.1 umol/L,0.5 umol/L,1 umol/L,5 umol/L,10 umol/L,20 umol/L,40 umol/L and 80 umol/L;Res 0.1,ResO.5,Res 1,Res 5,Res 10,Res 20,Res 40,Res 80)and blank group.(2)To determine whether the Shh signaling pathway plays a role in the effect of resveratrol on viability of NIH3T3 cells,five groups were studied:control group(Ctrl),resveratrol group(Res1),Shh signaling pathway Smo receptor inhibitor cyclopamine group(Cyc),resveratrol+cyclopamine group(Res 1 +Cyc)and blank group.(3)To examine whether Sirtl affects Shh signaling pathway,NIH3T3 cells were divided into six groups:control group(Ctrl),resveratrol group(Res1),Sirtl agonist SRT1720 group(SRT),Sirtl antagonist sirtinol group(Sirtinol),recombinant mouse sonic hedgehog group(Shh)and blank group.Cell viability was assessed by Cell Counting Kit 8(CCK-8).Immunocytochemistry assay was used to observe the nuclear translocation of Gli-1 protein and the Smo protein translocation into the primary cilia.The expressions of Shh signaling pathway proteins,which consists of Shh,Ptc-1,Smo,and Gli-1 proteins,were evaluated by Western blotting.Results:1.Concentration effects of resveratrol on viability of NIH3T3 cellsCCK-8 assay showed that the cell viability of the 0.5 and 1 ?mol/L resveratrol group(0.679±0.047,0.774±0.054)was significantly increased than those in the control group(0.585 ± 0.039)(P<0.01,P<0.001),and the highest viability was in the 1 ?mol/L resveratrol group.Along with the increase of resveratrol(10,20,40,and 80 ?mol/L),the cell viability gradually decreased(0.428 ± 0.043,0.395 ± 0.031,0.373 ± 0.017,0.361 ±0.016),and significantly decreased compared with the control group(P =0.000,P<0.001,P = 0.001,P = 0.001).However,there was no significant difference in the 0.1 ?mol/L(0.602 ± 0.065)and 5 ?mol/L group(0.556± 0.041)compared with the control group(P>0.05,P?0.05).Therefore,concentrations of 1?mol/L were selected for further study.2.Shh signaling mediates resveratrol to increase viability of NIHT3 cellsCCK-8 assay showed that compared with the control group(0.563±0.034),the viability of cells in the resveratrol group(0.778±0.371)was significantly increased(P<0.05).However,the effect of resveratrol on NIH3T3 cells viability was significantly adversed when cells were cultured with cyclopamine(5 ?mol/L)(0.439±0.259)alone or with cyclopamine combined with resveratrol(0.602±0.179).Furthermore,the minimum was in the cyclopamine alone group(P<0.05).Immunofluorescence assay showed that NIH3T3 cells have a primary cilium.In the control group,Smo lied in cytoplasm.In the resveratrol group,Smo translocated into the primary cilia.When NIH3T3 cells were cultured by cyclopamine alone or cyclopamine combined with resveratrol,Smo still deposited in cytoplasm.At the same time,Gli-1 in the control group accumulated in cytoplasm.In the resveratrol group,Gli-1 almost transferred to the nuclei from the cytoplasm.However,when cells were treated with cyclopamine alone or cyclopamine combined with resveratrol,Gli-1 was still situated in cytoplasm.Western blotting showed that expression of Gli-1 protein in nuclei,Shh,Ptc-1 and Smo protein in cytoplasm significantly increased in the resveratrol group than those in the control group(P<0.05).However,when cells were treated with cyclopamine alone or cyclopamine combined with resveratrol,the effects of resveratrol on the expression of Shh,Ptc-1,Smo and Gli-1 proteins were remarkably suppressed.Moreover,the suppressing effect of cyclopamine alone was strongest(P<0.05).3.Effects of Sirtl on Shh signaling in NIH3T3 cellsCCK-8 assay showed that resveratrol(0.751±0.05),SRT1720(0.793±0.037)and Shh protein(0.838±0.418)significantly strengthened the viability of NIH3T3 cells than those in the control group(0.531±0.034)(P<0.05).In contrast,Sirtinol(0.448±0.030)significantly decreased viability of the NIH3T3 cells than those in the control,resveratrol,SRT1720 and Shh protein groups(P<0.05).Immunofluorescence double labeling analysis showed that Smo accumulated in cytoplasm in the control group.Smo translocated to the primary cilia in the resveratrol,SRT1720 and Shh groups.On the contrary,Smo still lied in the cytoplasm in the Sirtinol group.Meanwhile,Gli-1 accumulated in cytoplasm in the control group.Gli-1 almost transferred to the nuclei from the cytoplasm in the resveratrol,SRT1720 and Shh groups.However,Gli-1 was still situated in cytoplasm in the Sirtinol group.Western blotting assay showed that the expressions of Shh,Ptc-1,Smo proteins in cytoplasm and Gli-1 protein in nuclei were significantly increased in the resveratrol,SRT1720 and Shh groups than those in the control group(P<0.05).However,the expressions of Shh,Ptc-1,Smo proteins in cytoplasm and Gli-1 protein in nuclei in the Sirtinol group significantly decreased compared with the control,resveratrol,SRT 1720 and Shh protein groups(P<0.05).Conclusion:Resveratrol could activate the Shh signaling to enhance viability of NIH3T3 cells and Sirtl may be a regulator for upstream of the Shh signaling pathway.