Attenuating Effect Of GSPE On Salmonella LPS-induced Inflammatory Injury Of Chicken Ovary

The extensive use of antibiotics in recent decades has led to development of serious antibiotic residues and antibiotic flora in animal products.Therefore,antibiotic replacement drugs with antibacterial efficacy are extensively studied.Natural antioxidant grape seed proanthocyanidin extract(GSPE)has attracted more and more attention because of its anti-inflammatory effect.It has potentials as an alternative to antibiotics.The clinical symptoms,histomorphology and gene expression levels were explored for the pathogenicity of Salmonella to 1-day-old chicks,and the effect of Salmonella LPS on chick development and its pathogenic mechanism were explored.Furthermore,the effect of proanthocyanidins on hen small yellow follicle granulosa cell damage induced by Salmonella LPS,was explored,along with its mechanism from cell proliferation,cell division,and TLR4/NF-κB signaling pathway.It will provide a theoretical basis for the rational development of anti-inflammatory antibacterial substances to prevent reproductive damage caused by bacterial endotoxin.1.Pathogenicity of Salmonella pullorum to chicks ovary and its mechanism.In this study,one-day-old SPF chicks were used.100 chicks were randomly divided into five groups and 500 μL of MY0259,MY00664,MY00682,and CVCC0534 Salmonella pullorum were fed orally at a concentration of 1.0×109 CFU/mL.The control chicks received the same dose of 0.9%physiological saline.After chicks were infected with Salmonella,the effects of several Salmonella strains on the life and health of chicks were evaluated from aspects of growth performance,overall mortality,clinical scores,and morphology.The qRT-PCR was used to detect gene expression of TLR4 receptors and inflammatory factors in the ovaries.The positive rate of CD 14 in the ovaries was detected by immunofluorescent staining.The results showed that the growth performance of the MY0259,MY00664,MY00682 and CVCC0534 groups were significantly decreased,compared with the control(P<0.05).The clinical performance of the chicks in the challenge group was poor.HE staining showed damage to the ileum villi and obvious lesions.The granulosa cells in the ovaries were abnormal,and the number of follicles were significantly rEdUced(P<0.05).the CD 14 positive rate in the ovarian cells were significantly elevated(P<0.05);LPS content in the ovary was significantly increased(P<0.05);Relative expression levels of TLR4 signaling pathway genes were significantly up-regulated(P<0.05).Therefore,Salmonella pullorum caused serious damage to chicks after infection and LPS was involved remarkably in this process.2.Relief of GSPE on inflammatory damage of ovarian granulosa cells induced by LPS of Salmonella.In the in vitro culture system of small yellow follicle granulosa cells,the toxic effect of LPS on granulosa cells was studied by detection of cell viability and observation of cell morphology.The results showed that after 6 h of LPS treatment of granulosa cells,the granulosa cells in the LPS(125 μg/mL)treatment group were damaged,the cells contracted,and the cell viability reached IC 50.Thereafter,GSPE(1,10,100 μmol/L)and LPS(125 μg/mL)were administered alone or in combination with GSPE for a total of 6 h.The results showed that the granulosa cells in the combination group with 10μmol/L GSPE and LPS were significantly protected as the pseudopods and the cell morphology were restored to normal and cell vibility was increased significantly(P<0.05).EdU and TUNEL tests were performed to detect cell proliferation and transformation.The results showed that the group treated with GSPE and LPS at the same time could significantly alleviate LPS-induced granulosa cells Apoptosis(P<0.05)and significantly increase granulosa cells proliferation(P<0.05).These results indicate that GSPE may alleviate reproductive damage induced by LPS.3.Exploration of the mechanism of GSPE and LPS on granulosa cells.In the in vitro culture system of small yellow follicle granulosa cells,GSPE(10μmol/L)and LPS(125 μg/mL)were separately or simultaneously administered for 6 h.Changes in the antioxidant level of granulosa cells were detected by antioxidant enzyme activity;TLR4/NF-κB signaling pathway and pro-inflammatory cytokine gene expression levels were detected by immunofluorescence staining and qRT-PCR.The results showed that the H2O2 level of the granulosa cells in the LPS(125 μg/mL)treatment group were increased significantly(P<0.05),and the GSH,T-SOD were decreased significantly(P<0.05),while the GSPE treatment group had relatively insignificant difference,indicating that GSPE could alleviate the oxidative damage of granulosa cells caused by LPS.The relative expression levels of TLR4,NF-κB,TNF-α,IL-1β,IL-6 and 1L-8 genes in the LPS treatment group were significantly increased(P<0.05),while the GSPE and LPS co-treatment groups were significantly decreased.(P<0.05).The positive rate of CD 14 in granulosa cells in the LPS treatment group was significantly increased(P<0.05),while it was significantly decreased in the GSPE and LPS co-treatment group(P<0.05).In summary,10 μmol/L GSPE may act on LPS-induced cytotoxicity through antioxidant and anti-effects.Among them,TLR4 signaling pathway in granulosa cells alleviate LPS induced damage procyanidins play an important role.It provides a theoretical basis for the rational development of anti-inflammatory antibacterial substances to prevent reproductive damage caused by bacterial endotoxin.