Protective effects and the mechanism of lipoic acid on cell model of Parkinson’s disease induced by lipopolysaccharide

doi:10.3969/j.issn.2095-4344.0595

Abstract

Abstract:

BACKGROUND: Neuroimmune and inflammatory reaction have been shown to participate in the occurrence of Parkinson’s disease. Lipoic acid possesses anti-inflammatory and neuroprotective roles.
OBJECTIVE: To investigate the protective effects of lipoic acid on PC12 cell models of Parkinson’s disease induced by lipopolysaccharide and the underlying mechanism.
METHODS: PC12 cells were divided into blank control, model (Parkinson’s disease model induced by lipopolysaccharide) and treatment (Parkinson’s disease model treated by lipoic acid) groups. PC12 cells were pretreated with lipoic acid for 2 hours, and then stimulated with lipopolysaccharide. The optimal concentration of lipopolysaccharide and lipoic acid was determined by cell counting-kit 8 assay. The expression of tyrosine hydroxylase was detected by immunohistochemistry. The levels of tumor necrosis factor α and interleukin 6 were detected by ELISA kit. The protein expression levels of nuclear factor-κBp65 and tyrosine hydroxylase were detected by western blot assay.
RESULTS AND CONCLUSION: The optimal concentration of lipopolysaccharide was 600 μg/L for constructing cell model of Parkinson disease, and 25 μmol/L lipoic acid was the optimal intervention concentration. Lipoic acid could increase the number of tyrosine hydroxylase positive cells, reduce the release of inflammatory factors in cell model of Parkinson’s disease, inhibit the production of nuclear factor-κBp65 and increase tyrosine hydroxylase content. Therefore, lipoic acid exhibits its effects on cell model of Parkinson’s disease through inhibiting the nuclear factor-κB signaling pathway and reducing inflammation, thereby exerting neuroprotection.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Thioctic Acid, Parkinson Disease, Lipopolysaccharides, Interleukin-6, Tumor Necrosis Factor-alpha, Tissue Engineering

CLC Number:

R446

Gao Hua, Li Yanxia, Wang Dan, Yang Xinling. Protective effects and the mechanism of lipoic acid on cell model of Parkinson’s disease induced by lipopolysaccharide[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(3): 447-452.

Figures/Tables 1

2.1 脂多糖诱导的帕金森病细胞模型的建立在200，600，1 000μg/L不同脂多糖质量浓度下，其细胞存活率分别为85%，49%，30%，相对于未进行干预的细胞组为基准(100%，单纯加入CCK8检测)。通过CCK8方法筛选建立帕金森病细胞模型的最合适的脂多糖质量浓度为 600 μg/L，见图1，在此质量浓度下细胞的存活率在50%左右，故作为后续实验研究条件。 2.2 硫辛酸干预细胞模型的最佳浓度的测定在确定了合适的脂多糖(600 μg/L)建造帕金森细胞模型的质量浓度后，在脂多糖-帕金森病模型之前给予硫辛酸进行保护性干预2 h后，在不同浓度(1，25和 50 μmol/L)采用CCK8方法检测其细胞存活率分别为51%，71%，61%，以未进行干预的细胞组为基准(100%，单纯加入CCK8检测)，最合适的硫辛酸的浓度为25 μmol/L，在此浓度下，相对于 1 μmol/L和50 μmol/L，对细胞存活率有很好的保护作用，可以使单纯的脂多糖-帕金森病组细胞存活率从51%上升至71%，以此为最佳浓度，见图2。 2.3 各组细胞酪氨酸羟化酶的表达可以看到表现胞浆为棕黄色的酪氨酸羟化酶的表达，细胞胞体略有缩小，突触更明显。PC12的细胞正常组中，酪氨酸羟化酶的阳性细胞数表现最为多。在给予脂多糖(600μg/L)建造的帕金森病细胞模型中，可以发现细胞活性部分被损害，脂多糖-帕金森病组中酪氨酸羟化酶的阳性细胞数减少，给予硫辛酸(25 μmol)保护性干预后可以观察到酪氨酸羟化酶的数量略有所恢复，将3组(每组包含有15个视野)的酪氨酸羟化酶阳性细胞数进行比较[(143±4)，105±5)，(124±1)个]，差异有显著性意义(P < 0.05)，见图3，4。 2.4 ELISA法检测各组细胞炎症因子表达在正常细胞组、脂多糖-帕金森病组和硫辛酸+脂多糖-帕金森病组的3组细胞中，白细胞介素6和肿瘤坏死因子α水平在脂多糖-帕金森病组相对于正常细胞组明显增加，加入硫辛酸后可以抑制两者的产生，从而使其表达有所下降。即白细胞介素6和肿瘤坏死因子α水平在脂多糖+硫辛酸组中，相比较脂多糖-帕金森病组有所减少，但较正常细胞组仍有较高表达[白细胞介素6：(80.02±1.25)，(138.10±2.11)，(109.60±1.30) ng/L；肿瘤坏死因子α：(116.60±3.55)，(228.00±2.12)，(162.00±5.08) ng/L]，3组间比较差异均有显著性意义(P < 0.05)，见图5。 2.5 Western blot检测各组NF-KBP65和酪氨酸羟化酶的表达正常细胞的对照组中，酪氨酸羟化酶和NF-κB p65均有表达。以此为对照基线，在脂多糖-帕金森病组中NF-κB p65表达含量增加，同时促使酪氨酸羟化酶表达含量减少。给予硫辛酸干预后可降低脂多糖-帕金森病组中NF-κB p65的表达含量，提高的酪氨酸羟化酶的表达。但硫辛酸-脂多糖-帕金森病组中两者相对于正常细胞的对照组中，不能恢复正常状态。其两者的含量在3组比较差异均有显著性意义(P < 0.05)，见图6。"

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