Objective To explore the effect and mechanism of ligustrazine on proliferation and apoptosis of inflammatory cell models in Parkinson's disease.Methods Human microglial cells(HMC3)in logarithmic growth phase were randomly divided into control group,lipopolysaccharide(LPS)group,LPS+12.5 μmol·L-1 ligustrazine group,LPS+25.0 μmol·L-1 ligustrazine group,LPS+50.0 μmol·L-1 ligustrazine group,and LPS+100.0 μmol·L-1 ligustrazine group.The cells in the control group did not receive any drug intervention.The cells in the LPS group,LPS+12.5 μmol·L-1 ligustrazine group,LPS+25.0 μmol·L-1 ligustrazine group,LPS+50.0 μmol·L-1 ligustrazine group,and LPS+100.0 μmol·L-1 ligustrazine group were first stimulated with 1 mg·L-1 LPS for 24 hours;the cells in the LPS+12.5 μmol·L-1 ligustrazine group,LPS+25.0 µmol·L-1 ligustrazine group,LPS+50.0 μmol·L-1 ligustrazine group,and LPS+100.0 μmol·L-1 ligustrazine group were treated with 12.5,25.0,50.0,and 100.0 μmol·L-1 ligustrazine for 24 hours,respectively.The viability of cells in each group was detected by using the live cell counting kit-8,and the optimal concentration of ligustrazine was selected to intervene in cell viability.The HMC3 cells in logarithmic growth phase were randomly divided into control group,LPS group,LPS+mimic NC group,LPS+miR-145 mimic group,LPS+inhibitor NC group,LPS+miR-145 inhibitor group,ligustrazine+LPS group,ligustrazine+LPS+miR-145 mimic group,and ligustrazine+LPS+miR-145 inhibitor group.The cells in the control group were not intervened with any drugs;the cells in the LPS group were stimulated with 1 mg·L-1 LPS for 24 hours;the cells in the LPS+mimic NC group,LPS+miR-145 mimic group,LPS+inhibitor NC group,and LPS+miR-145 inhibitor group were transfected with mimic NC,miR-145 mimic,inhibitor NC,and miR-145 inhibitor by liposomal 2000 reagent,respectively,and then were stimulated with 1 mg·L-1 LPS for 24 hours;the cells in the ligustrazine+LPS group were first stimulated with 1 mg·L-1 LPS for 24 hours and then were intervened with 100.0 μmol·L-1 ligustrazine;the cells in the ligustrazine+LPS+miR-145 mimic group and ligustrazine+LPS+miR-145 inhibitor group were first transfected with miR-145 mimic and miR-145 inhibitor by liposomal 2000 reagent,respectively,and then were treated with 1 mg·L-1 LPS and 100.0 µmol·L-1 ligustrazine for 24 hours,respectively.The relative expression level of miR-145 in cells in each group was detected by using the real-time fluorescence quantitative polymerase chain reaction,the cell proliferation in each group was detected by using the 5-ethynyl-2'-deoxyuridine assay,the cell apoptosis in each group was detected by using the Hoechst 33258 staining assay,the levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-1 β and IL-6 in the supernatant of cells in each group were detected by using the enzyme-linked immunosorbent assay,and the expression levels of phosphatidylinositol 3-kinase(PI3K),protein kinase B(AKT),phosphorylated PI3K(p-PI3K),phosphorylated AKT(p-AKT),Cyclin D1 and caspase-3 proteins in cells in each group were detected by using Western blot assay.Results The viability of HMC3 cells in the LPS group,LPS+12.5 μmol·L-1 ligustrazine group,LPS+25.0 μmol·L-1 ligustrazine group,LPS+50.0 μmol·L-1 ligustrazine group,and LPS+100.0 μmol·L-1 ligustrazine group was significantly lower than that in the control group,and the viability of HMC3 cells in the LPS+25.0 μmol·L-1 ligustrazine group,LPS+50.0 μmol·L-1 ligustrazine group,and LPS+100.0 μmol·L-1 ligustrazine group was significantly higher than that in the LPS group(P＜0.05).The viability of HMC3 cells in the LPS+12.5 μmol·L-1 ligustrazine group,LPS+25.0 μmol·L-1 ligustrazine group,LPS+50.0 µmol·L-1 ligustrazine group,and LPS+100.0 μmol·L-1 ligustrazine group significantly increased with the increase of ligustrazine concentration(P＜0.05).The concentration of 100.0 μmol·L-1 was taken as the concentration of ligustrazine in subsequent experiment.The relative expression level of miR-145 in HMC3 cells and the proliferation rate of HMC3 cells in the LPS group,LPS+mimic NC group,LPS+miR-145 mimic group,LPS+inhibitor NC group,and LPS+miR-145 inhibitor group were significantly lower than those in the control group,while the cell apoptosis rate was significantly higher than that in the control group(P＜0.05).The relative expression level of miR-145 in HMC3 cells and the proliferation rate of HMC3 cells in the LPS+miR-145 mimic group were significantly higher than those in the LPS group,LPS+mimic NC group,LPS+inhibitor NC group,and LPS+miR-145 inhibitor group,while the cell apoptosis rate was significantly lower than that in the LPS group,LPS+mimic NC group,LPS+inhibitor NC group,and LPS+miR-145 inhibitor group(P＜0.05).The relative expression level of miR-145 in HMC3 cells and the proliferation rate of HMC3 cells in the LPS+miR-145 inhibitor group were significantly lower than those in the LPS group,LPS+mimic NC group,and LPS+inhibitor NC group,while the cell apoptosis rate was significantly higher than that in the LPS group,LPS+mimic NC group,and LPS+inhibitor NC group(P＜0.05).There was no statistically significant difference in the relative expression level of miR-145 in HMC3 cells and the proliferation and apoptosis rates of HMC3 cells among the LPS group,LPS+mimic NC group,and LPS+inhibitor NC group(F=1.926,0.181,0.520;P＞0.05).The relative expression level of miR-145 in HMC3 cells and the proliferation rate of HMC3 cells in the control group and ligustrazine+LPS+miR-145 mimic group were significantly higher than those in the LPS group,ligustrazine+LPS group,and ligustrazine+LPS+miR-145 inhibitor group,while the apoptosis rate of HMC3 cells was significantly lower than that in the LPS group,ligustrazine+LPS group,and ligustrazine+LPS+miR-145 inhibitor group(P＜0.05).There was no statistically significant difference in the relative expression level of miR-145 in HMC3 cells and the proliferation and apoptosis rates of HMC3 cells between the control group and the ligustrazine+LPS+miR-145 mimic group(P＞0.05).The relative expression level of miR-145 in HMC3 cells and the proliferation rate of HMC3 cells in the LPS group and LPS+miR-145 inhibitor group were significantly lower than those in the ligustrazine+LPS group,while the apoptosis rate of HMC3 cells was significantly higher than that in the ligustrazine+LPS group(P＜0.05).There was no statistically significant difference in the relative expression level of miR-145 in HMC3 cells and the proliferation and apoptosis rates of HMC3 cells between the LPS group and the ligustrazine+LPS+miR-145 inhibitor group(P＞0.05).The levels of TNF-α,IL-1β and IL-6 in the supernatant of HMC3 cells in the control group and ligustrazine+LPS+miR-145 mimic group were significantly lower than those in the LPS group,ligustrazine+LPS group,and ligustrazine+LPS+miR-145 inhibitor group(P＜0.05).There was no statistically significant difference in the levels of TNF-α,IL-1β and IL-6 in the supernatant of HMC3 cells between the control group and the ligustrazine+LPS+miR-145 mimic group(P＞0.05).The levels of TNF-α,IL-1β and IL-6 in the supernatant of HMC3 cells in the LPS group and LPS+miR-145 inhibitor group were significantly higher than those in the ligustrazine+LPS group(P＜0.05).There was no statistically significant difference in the levels of TNF-α,IL-1β and IL-6 in the supernatant of HMC3 cells between the LPS group and the ligustrazine+LPS+miR-145 inhibitor group(P＞0.05).The relative expression levels of p-PI3K,p-AKT,and Cyclin D1 proteins in HMC3 cells in the control group and ligustrazine+LPS+miR-145 mimic group were significantly higher than those in the LPS group,ligustrazine+LPS group,and ligustrazine+LPS+miR-145 inhibitor group,while the relative expression level of caspase-3 protein was significantly lower than that in the LPS group,ligustrazine+LPS group,and ligustrazine+LPS+miR-145 inhibitor group(P＜0.05).There was no statistically significant difference in the relative expression levels of p-PI3K,p-AKT,Cyclin D1,and caspase-3 proteins in HMC3 cells between the control group and the ligustrazine+LPS+miR-145 mimic group(P＞0.05).The relative expression levels of p-PI3K,p-AKT,and Cyclin D1 proteins in HMC3 cells in the LPS group and LPS+miR-145 inhibitor group were significantly lower than those in the ligustrazine+LPS group,while the relative expression level of caspase-3 protein was significantly higher than that in the ligustrazine+LPS group(P＜0.05).There was no statistically significant difference in the relative expression levels of p-PI3K,p-AKT,Cyclin D1,and caspase-3 proteins in HMC3 cells between the LPS group and the ligustrazine+LPS+miR-145 inhibitor group(P＞0.05).Conclusion Ligustrazine can activate the PI3K/AKT pathway and reduce the levels of inflammatory factors by upregulating miR-145 expression in LPS-induced HMC3 cells,thereby promoting cell proliferation and inhibiting cell apoptosis and inflammatory response.
Parkinson's diseaseligustrazinemicroRNA-145inflammatory cellcell proliferationcell apoptosisphos-phatidylinositol 3-kinaseprotein kinase B
维普
万方数据
