Intervention And Molecular Mechanism Of Hydroxy Safflower A On Inflammatory Injury In Bovine Endometrial Epithelial Cells Induced By LPS

Cow endometritis is the main cause of infertility in dairy cows,which can increase the number of artificial insemination,breeding and extension the interval of pregnancy,reduce the rate of pregnancy and pregnancy,etc.Pathogenic bacteria are one of the main causes of endometritis in dairy cows,that E.coli is the most important.E.coli can produce a large amount of LPS,it can cause severe inflammation on the endometrium.Traditional Chinese medicine can prevent cow endometritis effectively,Safflower is a traditional Chinese medicine,it has the function of promoting blood circulation,removing blood stasis and relieving pain,which is commonly used in the treatment of endometritis in dairy cows.The hydroxy safflower yellow A is the main water-soluble active ingredient of Carthamus tinctorius,Therefore,to investigate the effect of HSYA and its molecular mechanism on inflammatory injury induced by LPS in bovine endometrial epithelial cells,the following studies were carried out:1.The endometrial epithelial cells were isolated with typsin and culturedin vitro..Then the cells were purified by differential digestion,Finally,the endometrial epithelial cells were identified by immunohistochemistry.The method of isolation and culture of endometrial epithelial cells was established successfully,which laid the foundation for further study.2.The effects of different concentrations of LPS on the proliferation of endometrial epithelial cells in vitro were detected by MTT,The results showed that the effect of 1?g/m L LPS on cell proliferation was the most obvious after the effect of 24 hours(P<0.05).The results showed that 1?g/m L LPS could increase IL-1? and TNF-? secretion in cow endometrial epithelial cells in significantly.The results of ELISA showed that the IL-1? and TNF-? increased significantly in bovine endometrial epithelial cells after treatment with LPS(P<0.05).Finally,the concentration of LPS was 1 ?g/m L,and the time was 24 h.3.The endometrial epithelial cells were treated with 1?g/m L,5.0?g/m L,10.0?g/m L,20.0?g/m L,40.0?g/m L,80.0?g/m L of HSYA for 24 h,Then it was used to detect the effect of HSYA on the proliferation of bovine uterine epithelial cells by MTT.The results showed that HSYA had no obvious toxicity on endometrial epithelial cells,and it could promote proliferation.Therefore,the concentration of HSYA was determined to be 80 ?g/m L(high dose group),20 ?g/m L(medium dose group),and 5 ?g/m L(low dose group).4.The endometrial epithelial cells of cows were treated with high,medium and low doses of HSYA after 24 h,Then method of real-time PCR was used to detect the expression levels of TLR4,IL-1?,IL-6,IL-8 and TNF-? m RNA in epithelial cells of.The results showed that the expression of TLR4 and cytokines IL-1,1L-6,IL-8,LPS,TNF-? m RNA were significantly higher than those of control group in endometrial epithelial cells of dairy cows(P<0.05).The expression levels of TLR4 and TNF-?,IL-1?,IL-8 and 1L-6 were significantly decreased(P<0.05)after 24 hours of 5,20,80 ?g/m L HSYA treatment in each group.The results showed that HSYA played an anti-inflammatory effect,it could inhibit the expression of TLR4 and inflammatory cytokines m RNA in the endometrial epithelial cells of dairy cows,which were induced by LPS.5.Western-blot was used to detect the expression of key proteins p-I?B?/I?B?,p-p65/p65 and proteins of P-JNK,p-p P38,p-ERK1/ERK2 in NF-?B and MAPKs of bovine endometrial epithelial cells.It was used to determine the role of HYSA in the cellular signaling pathway which induced inflammatory lesions in the endometrial epithelial cells of dairy cows by LPS,and find out the molecular mechanism.The results show that LPS can stimulate the ratio of p-I?B?/I?B? and p-p65/p65 increasing significantly(P<0.05)).At the same time,the expression levels of p-JNK,p-ERK1/2 and p-p38 were increased in significantly.That LPS can activate NF-?B and MAPK,leading to cow endometrial epithelial cells inflammatory injury;After the high,middle and low dose of HSYA,the ratio of p-I?B?/I?B? and p-p65/p65 significantly was decreased significantly(P<0.05)in cow endometrial epithelial cell.Compared with LPS group,the difference of p38 phosphorylation water was not significant(P>0.05),except for low dose HSYA group.The phosphorylation of JNK,ERK1/2 and P38 were decreased in significantly(P<0.05)after treatment with middle and high dose of HSYA.Therefore,the results show that HSYA can prevent the expression and secretion of cytokines to achieve the action of anti-inflammatory by relying on inhibition of NF-?B and MAPK signal pathway.