Experiemental Study On The Inhibition Of Colitis By Anhua Brick Tea Extract In Mice

Anhua black tea is produced by fresh leaves of Hunan xuefeng mountain tea varieties.After the four big processes of collecting,rolling,heap,pine wood fire drying,it becomes color black,brown oil moisten,elegant taste or micro astringent,bright tea solution,which has a unique aromatic smell and high quality characteristics.It has been shown that black tea has many health functions and is beneficial to the human body.These physiological health benefits are mainly brought by tea polyphenols,tea polysaccharide and tea pigment.Black tea physiological health functions includes antioxidant,removal of oxygen free radicals,lowering-lipid and regulating lipid metabolism,hypoglycemic,regulating sugar metabolism and protecting cardiovascular,antitumor,antibacterial antibacterial function.Recent studies have been reported that the extract of the tea leaves may also have a physiological anti-inflammatory effect,but the mechanism is not clear and it is still in the primary stage of research.Inflammatory bowel disease(IBD)includes various intestinal inflammatory diseases,usually IBD is damaged the ileum and rectum and colon,which results in inflammatory bowel disease.IBD is commonly appeared diarrhoea,abdominal pain.and even some people can appear the symptom of blood.Inflammatory bowel disease mainly includes two types:ulcerative colitis(ulcerative colitis,UC)and Crohn's disease.UC is a kind of inflammation and damages the colon and rectum,which can be associated with ulcer disease,but its specific etiology is not clear.Recent studies indicated that black tea may have anti-inflammation functions,but it is molecular mechanism is unclear.The expression level of mouse colitis factor mRNA was analyzed by RT-qPCR technique.The results showed that relative expression level of inflammatory factors TNF-a,IL-1 beta and IFN-gamma mRNA in the colon tissue of group DSS mice increased significantly compared with the control group,but the inflammatory factors in the colon of the mice of Anhua black tea extraction group were significantly inhibited.The inhibitory effect of Anhua black tea extract on the colitis of ICR mice induced by DSSThrough continuous drinking of 3%DSS 7 d,ICR mice were induced to establish ulcerative colitis model,and the anti-inflammatory effect of Anhua black tea extract on colitis was studied.During the process of experiment,mouse weight loss was observed in DSS group mice,the hair color of mice was became dim and appeared depression and severe diarrhea symptoms;besides this,the feces of some mice were observed bloody mucus by naked eye.Compared with the control group mice,DAI score of DSS group mice were increased significantly(p<0.01).Compared with the DSS group mice,high and low dosages of Anhua black tea treatments obviously ameliorated DSS-induced symptom in mice.Anhua black tea treatments reduce DSS-induced body weight loss,the incidence of fecal occult blood and diarrhea.The protective effect of high dosage Anhua black tea extract group is more obvious.After the experiment,all mice were killed and dissected with dislocated method.The colon of mice was taken out completely,and the results were observed and measured.The results showed that the intestinal wall of the DSS group was thickened obviously,and the intestinal mucosa had serious congestion and edema;Compared with the control group,the colon length was significantly shortened(p<0.01),and then the mice colon tissue was added to the phosphate buffer solution(phosphate buffer,PBS)homogenate,and the biochemical indexes in the homogenate supernatant were measured.The results showed that the activity of myeloperoxidase(myeloperoxidase,MPO)and malondialdehyde(malonaldehyde,MDA)increased significantly compared to the control group,while the concentration of MPO and malondialdehyde(MDA)in the two concentration groups of the black tea extract of Anhua black tea decreased significantly(p<0.01).Meanwhile,Anhua black tea extract had no significant thickening of colon wall and effective improvement of colonic mucosal congestion.The experiment shows that Anhua black tea can improve the immune system disorder in ulcerative colitis mice,reduce the content of MPO and MPO in the colon tissue,and reduce the damage of the colitis tissue in mice.The expression level of inflammatory factor protein in mouse colon was analyzed by Western blot.The results were consistent with those of RT-q PCR.The expression of inflammatory factors in colonic tissue of Anhua black tea extraction group was determined by immunohistochemical technique,suggesting that Anhua black tea could inhibit the expression of inflammatory factors in DSS induced colitis.RT-qPCR technique was used to analyze the mRNA expression levels of colitis factors in mice.The results showed that DSS treatment significantly increased mRNA expression levels of inflammatory factors TNF-?,IL-1? and IFN-y in the colonic tissue of DSS group mice compared with the control group,while Anhua black tea extraction treatments reduced the inflammatory factor expressions in the colonic tissues.Western blot technique was used to analyze the protein expression levels of inflammatory factors in the colonic tissues,and found that the protein expression level was basically consistent with the results of RT-qPCR technology analysis.The expression of inflammatory factors in colonic tissue was determined by immunohistochemical technique,and further confirmed that Anhua black tea extraction could inhibit the expression of inflammatory cytokines in DSS induced colitis in mice.Anhua black tea inhibits LPS induced inflammation and expression of inflammatory factors in macrophagesThe macrophage RAW264.7,stimulated by lipopolysaccharide(LPS),can be induced as an in vitro model of inflammatory factors,and to observe the inhibitory effect on the inflammatory state by using Anhua black tea.The effect of Anhua black tea on LPS induced RAW264.7 cytotoxicity.After 24 h of different concentrations of Anhua black tea were treated with 24 h cells,the cells were observed under the optical microscope,and the number of cells in each group was dense,the shape was spindle shaped,the growth state was good,and there was no obvious difference in the cell state.MTS method was used to detect the effect of Anhua black tea extract on RAW264.7 cytotoxicity.Between the control group and the Anhua tea group OD value had no significant difference(p>0.05).The experiment showed that in the range of 0?100 ?g/mL Anhua black tea extract had no obvious cytotoxicity on RAW264.7 cells,and had no effect on its growth,state and activity,and could be used for subsequent experimental research.The effect of Anhua black tea on the expression of inflammatory factors induced by LPS in RAW264.7 cells.RT-qPCR and Western blot were used to compare the expression of inflammatory genes in each group.Compared with the control group,the mRNA expression and protein expression of proinflammatory cytokines TNF a,IL-1 beta and IFN-gamma increased significantly.Compared to group LPS,Anhua black tea group could reduce the expression of these mRNA and protein,and the results showed a certain dose effect relationship.The effect of Anhua black tea on the NF-?B activity of LPS induced RAW264.7 cells.Nuclear transcription factor(nuclear transcription factor-?B,predominate the NF-?B p65/p50)is an important transcription factor,control inflammation factor expression of TNF-?,IL-1?,IFN-y gene promoter regions there are one or more of the nf-kappa B binding sites.The effect of anhua black tea on the activity of NF-?B was studied by using luciferase reporter gene technology.Compared with the control group,the relative fluorescence intensity(RLU)was significantly increased in the LPS induction group.However,the RLU value decreased significantly in the cells of anhua black tea extract group,and showed a dose dependence pattern,suggesting that the extract of anhua black tea may inhibit the expression of inflammatory factors by inhibiting the activity of NF-?B.To sum up,the extract of anhua black tea has inhibitory effect on the mouse colon inflammation induced by DSS,and it can significantly improve the inflammatory symptoms in mice.Anhua black tea extract can down-regulate the expression of key inflammatory cytokines TNF-?,IL-1? and IFN-?,and the molecular mechanism of its action may be related to the inhibition of NF-?B activity by Anhua black tea extract.