Regulatory Effects Of Osthol On Inflammation And Autophagy Of Bovine Mammary Epithelial Cells

Bovine mastitis has a high incidence in China’s breeding industry,which seriously restricts the development of the breeding industry.Lipopolysaccharide(LPS)in the cell wall of Gram-negative bacteria is a typical drug for the construction of inflammatory model,which can activate the signal pathway of cytokines and corresponding transcription factors,thus induce the body to produce inflammatory response.Autophagy plays an important role in maintaining the relative stability of intracellular environment.More and more studies have shown that many pure natural Chinese herbs can induce autophagy and autophagy can regulate the process of inflammation in many ways.Osthol(osthole,Ost),which comes from natural Chinese herbal medicine,has a variety of biological functions and plays an important role in regulating inflammatory response in vivo and in vitro,but its protective effect on inflammatory injury of dairy cow mammary epithelial cells and its regulatory mechanism on autophagy are not clear.Therefore,this experiment takes dairy cow mammary epithelial cells(MAC-T)as the research object,LPS as the modeling drug and osthol as the therapeutic drug to carry out the following studies:(1)Using CCK-8 method to screen and determine the best concentration of LPS;DAPI staining to detect the effect of different concentrations of LPS on the growth of MAC-T cells;qRT-PCR to detect the level of typical inflammatory cytokines to verify whether the cow mammary epithelial inflammatory model is constructed successfully.(2)The maximum nontoxic concentration of osthol was determined by CCK8 method,the effect of osthol on LPS-induced cell injury was detected by CCK-8 and DAPI staining.(3)To evaluate the effect of osthol on intracellular autophagy in MAC-T cells,acridine orange(AO)staining and(MDC)staining were used to detect the occurrence of autophagy and to evaluate the effect of osthol on intracellular autophagy;the relative expression of IL-1β、TNF-α and IL-6 mRNA in cells was detected by qRT-PCR,and the effect of osthol on the level of inflammatory factors in MAC-T cells induced by LPS was detected.The results showed that:(1)LPS of 0.1 mg/mL could significantly increase the survival rate of MAC-T cells(P<0.05),and the cell survival rate was about 90%,with the increase of LPS concentration,the cell survival rate decreased gradually and the cell injury was serious.After treatment with different concentrations of LPS,the number of cells decreased significantly,and the normal growth state of cells was affected.the mRNA expression levels of IL-1β、TNF-α and IL-6 in the model group were significantly increased(P<0.01).(2)15μM osthol had no significant effect on cell survival(P>0.05),When the concentration of osthol was more than 15 μM,the cell survival rate decreased significantly(P<0.01).Osthol pretreatment could inhibit the decrease of cell survival rate induced by LPS and reverse the change of cell growth state induced by LPS.(3)The results of AO staining showed that there were a lot of red fluorescence in the cytoplasm of different concentrations of osthol,and the red fluorescence spots increased gradually with the increase of osthol concentration,and the red fluorescence was the most in the 15 μM osthol treatment group.The results of MDC staining showed that there were obvious dot green fluorescence around the nucleus of 5 pM,10 μM and 15 μM osthol groups,and the fluorescence spots increased and the brightness increased with the increase of osthol concentration;The results of qRT-PCR showed that the mRNA expression levels of IL-1β、TNF-α and IL-6 in LPS treated group were significantly increased(P<0.01),osthol could significantly down-regulate the mRNA expression levels of IL-1β、TNF-α and IL-6(P<0.01).In conclusion,the best concentration of LPS to induce inflammation of bovine mammary epithelial cells is 0.1 mg/mL.In the LPS-induced inflammation model of mammary epithelial cells,osthol can significantly reduce the inflammatory injury caused by LPS,maintain the state of cell growth,improve the cell survival rate,and induce autophagy of MAC-T cells,down-regulate the mRNA expression of inflammatory factors IL-1β、TNFa and IL-6,thus playing an anti-inflammatory effect.