Protectice Effects And Mechanism Of L-theanine On Liver In Immune Stress Induced Mice

E. coli is one of the main pathogenic bacteria causing diarrhea and other tissues and organs infect. Liver is an important organ in the metabolism of substance and energy, and ETEC can induce the liver damage and liver function failure. The combination of antibiotics can cure the disease caused by the bacteria infection to a certain extent, but it is easy to produce drug resistance and toxic side effects. Therefore, the development and utilization of the plant functional components which safe, effective, not easy to produce drug resistance is an important development direction of the prevention and treatment of the infection of Escherichia coli. L-theanine is a characteristic amino acid in tea, which has many kinds of biological activities, such as antioxidation, regulating immunity and so on. The study showed that L-theanine had protective effect on alcoholic liver injury, but the protective effect on the bacterial infection liver injury was still to be further explored. For that reason, this study we used the SPF grade Balb/c female mice as experimental animals, after adaptive feeding 3ds, the mice were pre-treated orally with different dosages of L-theanine(100,300,500 mg·kg^-1) once daily for 30ds before ETEC E44813 intraperitoneal injection, sampling after 5hs, then from the angles of pathological changes, oxidative stress, inflammatory reaction, cell apoptosis and MAPK pathway to study the protective effect and its mechanism of L-theanine on the liver in immune stress mice induced by ETEC, to improve the liver health in modern diet structure and living environment, and provide scientific basis for the further utilization of L-theanine. The main research methods and results are as follows:（1） analyze the coefficient of liver and spleen, detedcted the levels of serum alanine aminotranferease （ALT） and aspartate aminotransferase （AST）, The pathological changes of liver tissue were observed by HE staining. studied the effect of L-theanine on the liver pathological changes of immune stress mice induced by ETEC. It turned out that pretreatment with L-theanine of different dosages significantly reduced the increase of liver and spleen coefficient caused by ETEC infection, lowered the levels of serum ALT, AST, and improved pathological injury of liver tissue.（2） Detected the liver tissue homogenate malondialdehyde （erythrocyte MDA） level and glutathione peroxidase （GSH PX）, catalase （CAT）, superoxide dismutase （SOD） activities, studied the effect of L-theanine on hepatic oxidative stress in mice with immune stress. It turned out that pretreatment with L-theanine of different dosages significantly reduced the increase of liver homogenate MDA caused by ETEC infection, improved the activity of liver homogenate SOD, CAT, GSH-Px.（3） Through analysed the expression of serum Tumor Necrosis Factor-a （TNF-a）, interleukin-1β (IL-1（3）, interferon gamma （IFN-y） and interleukin-4 （IL-4） by ELISA, and detected the mRNA expression of liver IFN-y, IL-4, TNF-α, IL-1β by real-time fluorescence quantitative polymerase chain reaction （PCR）, studied the effect of L-theanine on inflammatory reaction in liver of immunological stress mice. It turned out that pretreatment with L-theanine of different dosages significantly decreased the level of serum TNF-α、IL-1β、IFN-γ、IL-4, and the mRNA expression of IFN-γ、IL-4、TNF-α、IL-1β in liver homogenate。（4） Through detected the expression of Bcl-2, Bax mRNA and caspase-3 in liver by real-time fluorescence quantitative PCR and Western blot, to study the effect of L-theanine on the apoptosis of liver cells in immune stress mice. It turned out that pretreatment with L-theanine of different dosages significantly rised the mRNA expression of Bcl-2 and the ratio of Bcl-2/bax caused by ETEC infection, reduced the expression of Caspase-3 and Bax protein, and increased the expression of Bcl-2 protein.（5） Analized the phosphorylation of ERK1/2, JNK1/2 and p38 in liver by Western blot, from the level of signal pathway to explore the regulation mechanism of L-theanine on liver inflammation and apoptosis in immune stress mice, So as to preliminarily revealed the mechanism of L-theanine protected liver from ETEC infection in mice. Result show that pretreatment with L-theanine of different dosages significantly inhibited the phosphorylation of ERK1/2, JNK1/2 and p38 protein in mice liver induced by E.coli E44813 infection, inhibited the activation of MAPK signaling pathway.Conclusion:Pretreatment with L-theanine of different dosages can protect liver from Escherichia coli E44813 infection. And its mechanism of action is mainly by enhancing the antioxidant capacity of liver, reducing oxidative stress injury, inhibiting the expression of TNF-α、IL-1β、IFN-γ、IL-4 through inhibiting the activation of MAPKsignal pathway, alleviating inflammation, and meanwhile down-regulating the expression of Caspase-3, Bax protein, increasing the expression of Bcl-2 protein, inhibiting the apoptosis of liver cells.