The Effects Of ER Stress And Autophagy On The Apoptosis Induced By Microcystin-LR In Chinese Hamster Ovary Cells

Background and Objective Microcystins（MCs） are the most common algal toxin, which have more than 90 kinds of isomers. Microcystin-LR（MC-LR） is one of the most common and toxic isomers. MC-LR shows estrogen-like effect on fish and mammalian cells cultured in vitro, and may be an endocrine disrupting chemical. Endocrine disruptors could lead to disorder of hormone levels in the body and affect the normal breeding, growth and development of mammals.. With the advent of an aging society, the relationship between lower fertility and environmental pollution is attracted extensive attention of worldwide. The toxic effect of MC-LR on reproductive system and its toxicological mechanism have become a hot topic in environmental toxicology. However, the study on the damage of MCs induced reproductive system is mainly focused on male reproductive system, and its mechanism is mostly limited to the mitochondrial pathway at present. The mechanism of MCs toxicity on female reproductive system is not yet very clear involved the endoplasmic reticulum stress and autophagy signaling pathway. Further study on the mechanisms of the toxic effect of MC-LR on the reproductive system are important to protect the reproductive health, prevent and treat the disease of the reproductive system.Methods 1. CCK-8 kit was used to detect the proliferation inhibition rate of CHO cells after cells treated with different concentrations of MC-LR for 24 h; 2. Propyl iodide staining combined with flow cytometry were used to detect the cell cycle of CHO cells exposed to 0, 2.5, 5, 10 μM MC-LR; 3. DCFH-DA kit combined with flow cytometry were used to quantitatively detect the changes of the fluorescence intensity of reactive oxygen species（ROS） in the cells; 4. After exposed to 0, 2.5, 5, 10 μM MC-LR for 24 h, the content of calcium ion in CHO cells was detected by fluorescence probe Fluo3-AM with flow cytometry;5. The autophagosome was detected in CHO cells after exposed to MC-LR for 24 h by fluorescence probe monodansylcadaverine with live cells work station and flow cytometry; 6. After CHO cells were exposed to different concentrations of MC-LR for 24 h, Western blots was used to detect the expression of GRP78, ATF-6, IRE1, XBP1 and CHOP and LC3 B and Beclin1; 7. The changes of early apoptosis rate and late apoptosis rate of CHO cells were detected by Annexin V-PE/7-ADD staining using apoptotic rate kit and flow cytometry. 8. Statistical analysis was done with SPSS version 21.0 for windows. One-way analysis of variance（ANOVA） was used to analyze the difference between groups. Student-Newman-Keuls test（SNK） was used for multiple comparison in variances with homogeneity and Games-Howell test in variances without homogeneity. A value of P<0.05 was considered statistically significant.Results 1. Results of cell proliferation In the present study, the results according to MC-LR concentration and the times exposure to CHO cells showed that MC-LR could inhibit the proliferation of CHO cells. With the increasing of MC-LR concentration, cell proliferation was significantly inhibited compared to the control group（0 μM MC-LR）, and the difference was statistically significant（P<0.05）. The IC50 of MC-LR was 10 μM. Then 2.5, 5, 10 μM were selected as the exposure concentrations of MC-LR in follow-up experiments. 2. Results of cell cycle After CHO cells were incubated with 0, 2.5, 5, 10 μM MC-LR for 24 h, the number of cells in G₂/M had a significantly increase with the increasing of MC-LR, compared the control group（P<0.05）, which indicated that MC-LR could induce a disorder of cell cycle and thus decrsease cell proliferation rate. 3. Results of ROS The results suggested that the intracellular fluorescence intensity increased with the increasing of the exposure concentration. It also reflected that ROS content had an increasing trend in CHO cells, and the difference was statistically significant（P<0.05）. 4. Results of intracellular calcium The results revealed that with the increasing of the exposure concentration, calcium fluorescence intensity gradually increased（P<0.05）. When NAC was added to culture medium earlier 1 h than MC-LR, the calcium fluorescence intensity gradually increased with the increasing of the exposure concentration, but compared with the MC-LR group, calcium fluorescence intensity significantly decreased in NAC plus MC-LR group, and the difference was statistically significant（P<0.05）. 5. Results of autophagosome The results showed that with the increasing of MC-LR concentration, the number and brightness of autophagosome increased gradually in the CHO cells. And the flow cytometry results revealed that with the increasing of MC-LR concentration, the number of autophagy in the cells gradually increased compared to the control group, and the difference was statistically significant（P<0.05）. 6. Results of western blot The results displayed that endoplasmic reticulum stress marker proteins of GRP78, ATF-6, PERK, IRE1 and CHOP expression increased, autophagy marker proteins of Beclin1 and LC3 II expression increased and LC3 I expression decreased（P<0.05）. In 10 μM MC-LR group, autophagy inhibitor 3-MA and endoplasmic reticulum stress inhibitor 4-PBA were added respectively in advance 1h. Autophagy inhibitor 3-MA can increase the expression of endoplasmic reticulum stress marker protein, and the endoplasmic reticulum stress inhibitor 4-PBA can reduce the expression of autophagy marker protein. The results showed that the occurrence of endoplasmic reticulum stress could promote the occurrence of autophagy in apoptosis of CHO cells induced by MC-LR, and autophagy could inhibit the occurrence of endoplasmic reticulum stress. 7. Results of apoptosis rate MC-LR could increase the rate of early and late apoptosis of CHO cells. Total apoptosis rate increased with the increasing of the exposure concentration, and compared with the control group（0 μM MC-LR）, the difference is statistically significant（P<0.05）. In 10 μM MC-LR group, endoplasmic reticulum stress inhibitor 4-PBA was added in advance 1 h, and the apoptosis rate of CHO cells decreased; meanwhile, the apoptosis rate of CHO cells increased with autophagy inhibitor 3-MA was added in advance 1h before treated with MC-LR（P<0.05）.Conclusion 1. MC-LR could inhibit the proliferation of CHO cells, arrest cell cycle in G₂/M phase, and indue cells apoptosis. 2. MC-LR could induce elevation of ROS and Ca²⁺ levels in CHO cells, causing the occurrence of endoplasmic reticulum stress. 3. The endoplasmic reticulum stress pathway promotes the occurrence of apoptosis, and autophagy as a protective mechanism can inhibit the occurrence of apoptosis. Endoplasmic reticulum stress and autophagy pathways play an important role in the apoptosis of CHO cells induced by MC-LR.