| Parkinson’s disease (PD) is the second largest neurodegenerative disease, the main pathological features of which are the selective loss of dopaminergic neurons and the presence of Lewy bodies. Dopaminergic neuronal loss would cause a series of motor and non-motor symptoms, severely impairing the health of the old. Pathogenesis of PD is extremely complex, and environmental and genetic factors are likely to cause this disease. Oxidative stress is considered as an important factor of neuron degeneration in PD, so drugs with antioxidant activity might play positive effects on the treatment of PD. Stichopus Japonicus polysaccharide (SJP) is a kind of important active ingredient in the body wall of Stichopus Japonicus. Previous studies have confirmed that SJP could inhibit hypoxia/reoxygenation injury in PC 12 cells, and the mechanism may be related with the antioxidant activity. In this study, we investigated the protective effects of SJP on SH-SY5Y cells damaged by 6-OHDA and explored its possible mechanism. The main contents were as follows:1. The protective effects of SJP on SH-SY5Y cells damaged by 6-OHDAFirst, we examined the cell viability by MTT assay, and found that 6-OHDA decreased cell viability, whereas SJP can restore cell survival. Then we used Hoechst33342/PI kit to stain the cells and found that 6-OHDA would promote apoptosis and necrosis, while SJP could inhibit that. Next, we used the LDH kit to detect the LDH activity of the supernatant and found that 6-OHDA increased the release of LDH, which could be inhibited by SJP pretreatment. Next, the ROS and SOD kits were used to detect the level of intracellular ROS generation and SOD activity. Then we found that 6-OHDA elevated the level of ROS and reduce the activity of SOD, which could be reversed by SJP. These results indicated that SJP could inhibit 6-OHDA-induced oxidative stress and cell death.2. The mechanism of effects of SJP on SH-SY5Y cells damaged by 6-OHDAFirst we used JC-1 kit to detect the changes in mitochondrial membrane potential, and the results showed that 6-OHDA reduced mitochondrial membrane potential, and SJP could increased the mitochondrial membrane potential. Then we used the Western Blot analysis to detect the levels of Bax, Bcl-2, cytochrome c, procaspase-3, cleaved caspase-3, procaspase-9 and cleaved caspase-9. The results showed 6-OHDA elevated the ratios of Bax/Bcl-2, cytoplasm cytochrome c/mitochondrial cytochrome c, cleaved caspase-3/procaspase-3 and cleaved caspase-9/procaspase-9, and SJP could reduce the four kinds of ratios. The results showed that SJP could inhibit mitochondrial apoptosis pathway. Then we used the Western Blot analysis to detect the levels of p-p38, p38, p-JNK, JNK, p-ERK, ERK, p-Akt and Akt, and the results showed that 6-OHDA elevated the ratios of p-p38/p38, p-JNK/JNK and p-ERK/ERK and reduced the ratio of p-Akt/Akt. SJP pretreatment could reduce the ratios of p-p38/p38 and p-JNK/JNK, and elevate p-Akt/Akt’s ratio, indicating that SJP could inhibit the activation of p38 MAPK and JNK pathway and inactivation of PI3K/Akt pathway. Then we used the p38, JNK, ERK and Akt inhibitors and Western Blot analysis to detect the protein expression of the mitochondrial apoptotic pathway. Results showed that p38 and JNK inhibitors could further promote the inhibition of mitochondrial apoptosis by SJP and Akt inhibitor could attenuate the effects of SJP.In this study, we investigated the protective effects of SJP on 6-OHDA-induced damage of SH-SY5Y cells from the cellular level, and further clarified the protective mechanism. This study provided new method and theoretical basis for the treatment of Parkinson’s disease, and provided evidences for development and utilization of SJP and other marine drugs. |
PDF Full Text Request