An Experimental Study On The Neuroprotective Mechanism Of Minocycline In Dopaminergic Neurons

Part I Inhibitory action of minocycline on lipopolysaccharide-inducedexpression of induced nitricoxide synthase in microglial cellsObjective To explore the inhibitory effect of minocycline on lipopolysaccharide-induced production of nitric oxide (NO) and expression of induced nitricoxide synthase in BV-2 microglial cells. Methods The BV-2 microglial cells were treated with LPS(100ng/ml) alone or with various concentrations(0, 0. 1, 1, 10, 100μmol/L, respectively) of minocycline. Accumulated NO was measured in the cell supernatant by enzyme method. INOS protein was assayed by immunohistochemistry staining. RT-PCR examined the expression of iNOS mRNA. Results The NO production and the expressions of iNOS protein and mRNA were significantly increased in LPS group, and were significantly higher than those of control group (P<0. 05, 0.01,0. 01, respectively). The pre-treatment of MC (10μmol/L) could significantly inhibit the NO production and the expressions of iNOS protein and mRNA induced by LPS. The NO production and the expressions of iNOS protein and mRNA of MC+LPS group were significantly lower than those of LPS group (P<0. 01, respedtively), but still significantly higher than those of control group(P<0.05, respedtively). Conclusion The pre-treatment of MC could significantly inhibit the NO production and expression of iNOS protein and raRNA induced by LPS in microglial cell. Part II Inhibitory action of minocycline on lipopolysaccharide-inducedexpression of tumor necrosis factor-alpha in microglial cellsObjective To explore the inhibitory effect of minocycline on lipopolysaccharide-induced expression of tumor necrosis factor-α (TNF-α) in BV-2 microglial cells. Methods The BV-2 microglial cells were treated with LPS (100ng/ml) alone or with various concentrations (0, 0. 1, 1, 10, 100μmol/L, respectively) of minocycline. TNF-α concentration in the cell supernatant and TNF-α protein in BV-2 cell were assayed by enzyme linked immunosorbent assay(ELISA) and immunohistochemistry staining respectively. RT-PCR examined the expression of TNF-α mRNA. Results The TNF-α concentration in the cell supernatant and the expressions of TNF-α protein and mRNA in BV-2 cell were significantly increased in LPS group, and were significantly higher than those of control group (P<0. 01, respectively). The pre-treatment of MC (10μmol/L) could significantly inhibit the TNF-α concentration and the expressions of TNF-α protein and mRNA induced by LPS. The TNF-α concentration and the expressions of TNF-α protein and mRNA of MC+LPS group were significantly lower than those of LPS group (P<0. 01, respedtively). The TNF-α concentration in the cell supernatant of MC+LPS group was same with control group (P>0. 05). But the expressions of TNF-α protein and mRNA of MC+LPS group were still significantly higher than those of control group(P<0. 05, respedtively). Conclusion The pre-treatment of MC could significantly inhibit the expression of TNF-α induced by LPS in microglial cell.Part III Minocycline inhabits P38 mitogen-activated protein kinasepathway to suppress lipopolysaccharide-induced activation of microgliaObjective To explore the relationship between suppressive effect of minocycline to lipoploysaccharide-induced activation of BV-2 cells with p38 mitogen-activated protein kinase (P38MAPK) pathway. Methods The BV-2 microglial cells were treated with LPS(100ng/ml) alone or with 10μmol/L of minocycline. Western-blot examined the expression of P38MAPK protein in BV-2 cells. Results There were no significant differences of total p38MAPK protein in all groups. The expression of phosphorylation of p38MAPK (p-p38MAPK) protein in control group and MC group were not observed. The expression of p-p38MAPK protein in LPS group was significantly increased and higher than that of control group (P<0. 01). The expression of p-p38MAPK protein in MC+LPS group was significantly lower than that of LPS group (P<0. 01), but still significantly higher than that of control group(P<0.01). Cnclusion MC might suppress activation of microglial cells induced by LPS through inhibiting the p38MAPK pathway. Part IV Protective effect of minocycline to the apoptosis ofPcl2 cells induced by MPP~+Objective To explore the protective effect of minocycline and investigate the relationship between Caspase-3 mRNA expression with protective effect of minocycline to the apoptosis of cell parkinsonism models induced by MPP~+. Methods Using PC12 cell as the apoptotic model of dopaminergic neurons, MC and MPP~+ were added into the culture of pcl2 cells, and using MTT to assay the cell viability and metabolism state; Using electrophoresis method to assay cell apoptosis, using flow cytometry FACS to assay the apoptosis ratio; using RT-PCR to assay the caspase-3 mRNA expression. Results At the MPP~+ concentration of 10μmol/L, the cell parkinsonism model of apoptosis was established. The pre-treatment of MC (100μmol/L) could significantly increase the pcl2 cell viability. The apoptosis ratio of MC+MPP~+ group was significantly lower than the apoptosis ratio of MPP~+ group, but was still significantly higher than the apoptosis ratio of control group. The caspase-3 mRNA expression of MC+MPP~+ group was significantly lower than that of MPP~+ group, but was still significantly higher than that of control group. Conclusion MC might protect the cell apoptosis induced by MPP~+ to some extent and down-regulate the mRNA expression of caspase-3 to protect the cell apoptosis induced by MPP~+.