Research On Effects Of LPS And Heat Stress On Porcine Granulosa Cells And The Regulating Mechanism Of HSP70

Endotoxin infection and heat stress are the two most important environmental factors that affect the reproductive performance of animals. The chemical nature of endotoxin is lipopolysaccharide(LPS). LPS and heat stress can disturb health of animals through various paths. In this study, we focused on the adverse impact on animal reproduction. It has been demonstrated that, LPS or heat stress could disturb in vivo reproductive endocrine axis, inhibited hormone secretion and normal oestrus, finally reducing the ovulation, conception rate, and even live birth rate. Follicle granulosa cell is a kind of highly differentiated somatic cell, having close affinity with oocytes, which plays an important role in animal reproductive physiology. But there is little evidence about the effects and mechanisms of LPS and heat stress on granulosa cells directly. Therefore, in this study, the effects of LPS and heat stress on cell morphology, proliferation, apoptosis, hormone secretion of in vitro cultured porcine granulosa cells model and preliminary acting path were studied.The optimum challage time of LPS and heat stress were 48 h and 3 h respectively. Cell morphological changes were analyzed by scanning electron microscopy, and the images were enlarged 1000×, 5000×, 10000× and 15000×. In this study, granulosa cell proliferation, apoptosis and secretion of estrogen was determined by CCK8, flow cytometry using Annexin V-FITC/PI apoptosis detection kit and ELISA respectively. Cell MAPKs proteins including ERK, Phospho ERK, JNK, Phospho JNK, p38 MAPK, Phospho p38 MAPK were analyzed by Western Blot. The expressions of genes associated with cell proliferation and cell cycle including FN1, IGF2, IGFBP2, Cyclin D1, Cyclin D2, P27 kip and hormone secretion P450 arom and FSHR were tested by qRT-PCR. While the expression of HSP70 gene was also detected at the same time. The study found that granulosa cells HSP70 was able to activate by both LPS and heat stress. It was the intersection of the two negative environmental factors. Then P450 arom, FSHR and HSP70 genes or protein were re-evaluated by small molecules inhibitor(VER 155008) and activator(STA-4783) respectively. Based on HSP70 gene overexpression, the quantity and location of phosphorylation Smad3 were analyzd by cell immunofluorescence assaysResults showed that cells were plumpy and had more pseudopodiums and microvillis than control group after LPS treatment. Cell proliferation and survival ratio were significantly promoted at LPS concentrations of 1000 ng?m L-1(P<0.05). On the contrary, cell apoptosis was inhibited. However, gene expression levels of FN1(P<0.01), IGF2, IGFBP2(P<0.05), Cyclin D1 and Cyclin D2(P<0.01) were up-regulated at 500 ng?m L-1 of LPS. However P27kip(P<0.01) which hindered cell cycle was inhibited. Cell MAPKs protein content, the total proteins content remained unchanged while phosphorylated proteins content were enhanced. Cells treated by heat stress were being pyknotic, lost pseudopodiums and microvillis. P450 arom and FSHR gene expressions were decreased by both LPS and heat stress. E2 secretion could be inhibited by LPS(P<0.01) at the same time. HSP70 gene(P<0.01) and protein expression were significantly upregulated by LPS and heat stress. HSP70 gene relative quantitative expression was up-regulated and protein was inhibited after HSP70 inhibitors(VER 155008) application. Meanwhile P450 arom and FSHR gene expressions were significant restored(P<0.01). On the contrary, HSP70 gene was activated significantly(P<0.01) and the relative quantitative expression increased more than 50 times when HSP70 activator(STA-4783) treated. And protein expression increased significantly. In P450 arom and FSHR gene expressions appeared significantly lower than before. Smad3 phosphorylation and nuclear translocation were inhibited by HSP70 overexpression at 3 h and 48 h for the fluorescence intensity of nucleus was dimmed. Phospho Smad3 protein degraded substantially at the same time.In conclusion, the above results showed that, granulosa cell proliferation could be promoted by LPS, while cell apoptosis was inhibited. Granulosa cells' function could be inhibited by both LPS and heat stress. The inhibition of granulosa cells' hormone secretion may be caused by HSP70, Smad3 phosphorylation and nuclear translocation which hindered by HSP70 overexpression was one of the paths.