Study On Screening And Effect Of Anti-Inflammatory Traditional Chinese Medicine Compound

Enteritis is fairly common in veterinary clinic,bacteria,virus,parasite and improper management can result in inflammatory bowel disease,clinical use of antibiotics and other anti-inflammatory medication enteritis easy to produce side effects,drug residues situation is very serious,not only affect the animal husbandry development,also may do harm to human health and the natural environment.Traditional Chinese medicine(TCM)is a precious natural resource in China.With various kinds,remarkable curative effects,extensive pharmacological effects and low toxic and side effects,TCM has gradually become the research object of new anti-inflammatory drugs.The experiment selected Clematidis Radix Et Rhizoma,Polygonum flaccidum Meissn,Pomegranate Rind,Geraniumwilfordii Maxim,Berberidis Radix,Gerbera anandria(Linn.),Callicarpa formosana Rolfe,Smilax scobinicaulis C.H.Wright,Mahonia bealei(Fort.)Carr,Polygonum chinense L.these ten kinds of traditional Chinese medicine,to explore the anti-inflammatory effects of these ten kinds of traditional Chinese medicine.finally,Berberidis Radix,Gerbera anandria(Linn.)and Clematidis Radix Et Rhizoma,which have better anti-inflammatory effect,were selected to form a prescription.By comparing the anti-inflammatory effects of four compounds with different combinations of single drug and three herbs,the anti-inflammatory effect of the compound composed of three herbs was selected to be better,and the anti-inflammatory effect of this compound was further studied in vitro,and it was found that the antiinflammatory effect of this compound was better in vitro.The compound of three traditional Chinese medicines,Berberidis Radix,Gerbera anandria(Linn.)and Clematidis Radix Et Rhizoma,has the effect of clearing away heat and dampness,passing through menstrual channels and relieving pain,and can be used to remove dysentery from dampness and heat.Therefore,this study explored the protective effect of Chinese traditional Chinese medicine compound on LPS-induced enteritis.In this experiment,firstly,a mononuclear macrophage inflammation response model induced by lipopolysaccharide(LPS)was established in RAW264.7 mice.Ten Chinese herbal medicines with anti-inflammatory effects were screened,and three Chinese herbal medicines with good anti-inflammatory effects,Berberidis Radix,Gerbera anandria(Linn.)and Clematidis Radix Et Rhizoma were selected for formulation.Secondly,the inflammatory model of RAW264.7 cells induced by LPS was established.By comparing the anti-inflammatory effects of different combinations of single Chinese medicine and three kinds of Chinese medicine,it is found that the anti-inflammatory effect of the three kinds of Chinese medicine is better,It was found that the anti-inflammatory effect of the three TCM compounds was better,and then the anti-inflammatory effect of the three TCM compounds in vitro was explored.Finally,LPS-induced mouse enteritis model was established to explore the protective effect of TCM compound on LPS-induced enteritis,so as to provide a new reference for the development of anti-inflammatory drugs.This study is divided into the following three experimental parts:Experiment Ⅰ Screening of Anti-inflammatory effects of single traditional Chinese Medicine In order to screen out the traditional Chinese medicine with good antiinflammatory effect,this study firstly induced RAW264.7 cells with different concentrations of LPS,and determined the optimal concentration of LPS induced RAW264.7 cells to establish an in vitro inflammatory response model by detecting the level of nitric oxide in the cell supernatant.Then,through cell safety test,three optimal safe concentrations were selected,and then the cells were pretreated with water extract of traditional Chinese medicine for 1 hour.RAW264.7 cells were induced with LPS and co-cultured for 24 hours.After 24 hours,the cell culture supernatant was taken,and the NO content was detected by Griess method.The results showed that the five herbs of Clematidis Radix Et Rhizoma,Smilax scobinicaulis C.H.Wright,Berberidis Radix,Gerbera anandria(Linn.)and Callicarpa formosana Rolfe had significant inhibitory effect on NO,and then the IL-6 content was detected by ELISA method.To compare the effects of five herbs of Clematidis Radix Et Rhizoma,Smilax scobinicaulis C.H.Wright,Berberidis Radix,Gerbera anandria(Linn.)and Callicarpa formosana Rolfe on IL-6 level of LPS-induced RAW264.7 cells.The results showed that 5μg/ m L LPS was the best concentration to induce RAW264.7 cells to establish an in vitro inflammatory response model.Three kinds of traditional Chinese medicines,Berberidis Radix,Gerbera anandria(Linn.)and Clematidis Radix Et Rhizoma,had the best anti-inflammatory effect in vitro,so these three kinds of traditional Chinese medicines were selected to make the prescription for the follow-up test.Experiment Ⅱ Study on Anti-inflammatory effect of traditional Chinese Medicine prescription in Vitro In order to screen out the traditional Chinese medicine compound with good anti-inflammatory effect and further study the anti-inflammatory effect of the screened compound in vitro,the experiment composed 4 compounds of three kinds of traditional Chinese medicine with equal proportion according to different combinations.Through the cell safety test,three optimal concentrations of each compound were selected,and RAW264.7 cells were induced by LPS and co-cultured for 24 hours.After 24 hours,the supernatant of cell culture was taken to detect the contents of NO and cell inflammatory factors TNF-α,IL-6 and IL-1β,and the anti-inflammatory effects of each single Chinese medicine and four compounds with different combinations of three Chinese medicines were compared.Then,the cells were pretreated with the water extract of the selected TCM compound for 1 hour,and then the RAW264.7 cells were induced by LPS and co-cultured for 24 hours.The contents of NO,PGE2 and inflammatory cytokines TNF-α,IL-6 and IL-1βwere detected 24 hours after cell culture supernatant.Fluorescence quantitative PCR was used to evaluate the effects of the selected TCM compounds on the gene expression levels of nitric oxide synthase(iNOS),TNF-α,IL-6 and IL-1β.The results showed that the compound composed of three herbs had good anti-inflammatory effect in vitro,and had good inhibitory effect on the release of NO and inflammatory factors TNF-α,IL-6 and IL-1β in RAW264.7cell induced by LPS.The anti-inflammatory effect of the selected compound was further studied in vitro.The results showed that the compound had a good inhibitory effect on the release of NO,PGE2 and inflammatory factors TNF-α,IL-6 and IL-1β in RAW264.7 cells induced by LPS.It can also down-regulate the expression of iNOS,TNF-α,IL-6 and IL-1βgenes in RAW264.7 macrophages induced by LPS.Experiment Ⅲ Study on the Protective effect of traditional Chinese Medicine prescription on LPS-induced Enteritis In order to explore the anti-inflammatory effect of traditional Chinese medicine compound in vivo,mice were intraperitoneally injected with different concentrations of LPS at first.By observing the overall state of mice,blood biochemical indexes and the levels of inflammatory factors in jejunum and ileum tissues were detected to determine the optimal concentration of LPS to induce mouse enteritis model.And use traditional Chinese medicine compound water extraction liquid preventive lavage for 7days,the last day of the abdominal injection of LPS,injection in mice after 6 hours overall state,excrement and urine,to evaluate disease activity index,detection of blood biochemical index,serum levels of inflammatory cytokines in mice,intestinal tissue inflammatory factor and HE staining to observe the mice small intestine and liver tissue morphology,To explore the protective effect of Chinese herbal compound on LPS induced enteritis model.The results showed that TCM compound at 200 mg/ m L,100 mg/ m L and 50 mg/ m L had a certain protective effect on LPS-induced enteritis model in mice,and the anti-inflammatory effect of TCM compound at 200 mg/ m L and 100 mg/ m L was better.