| Objective: To observe the protective effect and mechanism of baicalin on LPS-induced neuron injury.Methods:1.Observation on the effect of baicalin on neuronal injury induced by LPS in SD rats(1)Experimental protocol: SD rat were intracerebroventricular injection given with LPS 120 ?g and baicalin was given by gavage once a day for a week.Rats were divided into four groups: Control group(Cerebrospinal fluid + CMC-Na),LPS group(LPS + CMC-Na),Low dose group(LPS + baicalein 50 mg/kg),High dose group(LPS + baicalein 100 mg/kg),and 10 rats per group.(2)Observation indexes: The learning and memory function of SD rats was measured by Morris water maze.The morphology of hippocampus and cortex of SD rats was observed by HE.The PI3K/Akt/mTOR,p62.LC3B-II,Beclin1 protein expressions of hippocampus and cortical of SD rats were detected by Western blotting.2.The effect of baicalein on LPS-induced HT22 cell injury(1)Determination of the optimal concentration and treatment time of LPS for LPS-induced HT22 cell injury:Cell viability was measured by MTT assay,cell LDH leakage rate was detected by biochemical enzyme assay,cell apoptosis was detected by flow cytometry,and cell morphology was determined by light microscopy.(2)The protective effect of baicalein on LPS-induced HT22 cell injuryExperimental grouping: Control group,LPS group,LPS+different concentrations of baicalein groupObservation indexes: The effect of baicalein on the survival rate of normal HT22 cells was detected by MTT assay to observe the toxicity of baicalein.MTT assay and biochemical enzymatic assay were used to determine the effect of baicalein on LPS-induced HT22 cell survival rate and LDH leakage rate.To determine the optimal concentration of baicalin for the protection of LPS-induced HT22 cell injury,cell apoptosis was observed by flow cytometry and cell morphology was observed by light microscopy.(3)Effect of baicalein combined with rapamycin/wortmannin on LPS-induced HT22 cell injuryExperimental group: Control group,LPS group,LPS+Baicalein group,LPS+Baicalein+Rapamycin/Wortmannin group,LPS+Rapamycin/Wortmannin group.Observed indexes: MTT assay was used to detect the survival rate of HT22 cells,biochemical enzymatic assay was used to measure the LDH leakage rate of HT22 cells,and western blotting was used to detect the expressions of PI3K/Akt/mTOR,p62,LC3B-II and Beclin1 in HT22 cells.Results: 1.Compared with the Control group,the learning and memory function of rats was significantly decreased in the LPS group.The hippocampal CA1 and cortical neurons were disordered arrangement,cell shrinkage,nuclear pyknosis and nuclear hyperchromatism number increased significantly.The expressions of PI3 K,Akt,mTOR,p-mTOR,p-Akt and p62 protein were significantly increased,and the expressions of Beclin1 and LC3B-II protein were significantly decreased in hippocampus and cortex.Compared with LPS group,administration of baicalein can significantly improve the learning and memory function of rats,the injury of hippocampal CA1 and cortical neurons were significantly improved,hippocampus and cortex expressions of PI3 K,Akt,mTOR,p-mTOR,p-Akt and p62 protein were significantly decreased,and the expressions of Beclin1 and LC3B-II protein were significantly increased.2.Compared with the Control group,the survival rate of HT22 cells in the LPS group was significantly decreased,the LDH leakage rate was significantly increased,and the apoptosis rate was also increased.Compared with the LPS group,the cell survival rate in LPS+Baicalein group was significantly increased,the LDH leakage rate was significantly decreased,the apoptosis rate was decreased,and the cell morphology was also significantly improved.Compared with the LPS+Baicalein group,the survival rate of cells in LPS+Baicalein+Rapamycin group was significantly increased,the LDH leakage rate was significantly decreased.The survival rate of cells in LPS+Baicalein+Wortmannin group was significantly decreased,and the LDH leakage rate was significantly increased.3.Compared with the Control group,the expressions of PI3 K,Akt,mTOR,p-mTOR,p-Akt and p62 protein in LPS group were significantly increased,and the expressions of Beclin1 and LC3B-II protein were significantly decreased.Compared with LPS group,the expressions of PI3 K,Akt,mTOR,p-mTOR,p-Akt and p62 protein in LPS+Baicalein group was significantly decreased,and the expressions of Beclin1 and LC3B-II protein were significantly increased in the HT22 cell.Compared with LPS+Baicalein group,the expressions of PI3 K,Akt,p-Akt,p-mTOR and p62 protein in LPS+Baicalein+Rapamycin group were significantly decreased,and the expressions of LC3B-II and Beclin1 protein were significantly increased,While the expressions in LPS+Baicalein+Wortmannin group of PI3 K,p-mTOR,LC3B-II and Beclin1 protein were significantly decreased,and the expression of p62 protein was significantly increased.Conclusion: Baicalein can improve the learning and memory impairment and neuronal damage induced by LPS in rats,and can also improve the injury of HT22 cells induced by LPS.The mechanism may be related to the inhibition of PI3K/Akt/mTOR pathway to up-regulate of the neuronal autophagy. |
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