The Anti-inflammatory Effect And Innate Immune Mechanism Of A Traditional Chinese Drug Prescription "Puheyin"

Inflammation is a biological response to try to remove it when something harmful or irritating affects a part of organism body,which the classical signs of acute inflammation are pain, heat, redness, swelling, and loss of function. These clinical symptoms such as fever,swelling,pain are similar to these "hot" types of syndromes in Traditional Chinese Medicine(TCM) especially,which involve symptoms like high fever,sweating,Red tongue with yellow sticky coating,skin sores and so on.The theory of TCM holds that predominance of "yang" generating "heat", which was converted by the invasion of exogenous pathogenic factor. The principle of treatment inflammation were "clearing away heat and detoxification,activating blood and dissolving stasis,dredging meridians and preventing pain" in TCM clinic practice. Traditional Chinese drug with those above-mentioned functions,such as honeysuckle,forsythia,dandelion,which were regarded as the "anti-inflammatory traditional Chinese medicine".Good clinical treating effects has shown,the uncertainty of mechanism and prescription severely restricted the application of anti-inflammatory traditional Chinese medicine.Therefore,research for anti-inflammatory mechanism of anti-inflammatory traditional Chinese medicine with modern medicine and molecular biology theory and technique.This is the basis of the clicical application of anti-inflammatory traditional Chinese medicine in both medicine and veterinary medicine field.According to the therapeutic principle of TCM,traditional Chinese drug prescription "Puheyin" was proposed.Inflammation models were used to determine whether "Puheyin" could decrease inflammatory reaction.The anti-inflammatory effect and innate immune mechanism of "Puheyin"were studied by the histologic method,Real-time Quantitative RT-PCR method and Enzyme-Linked Immuno Sorbent Assay method,in order to clarify the anti-inflammatory target of "Puheyin",and provide a basis for further utilizing.1Anti-Inflammatory effect of "Puheyin" in miceThe inhibitory effect on inflammation of traditional Chinese drug prescription "Puheyin"will be researched.Inflammation models,auricle swelling induced by dimethylbenzene and toes swelling caused by10%fresh albumen in mice,were established.Also,the optimal dose was determined.The results showed that Traditional chinese drug prescription "Puheyin" can significantly reduce auricle swelling rate and degree of swelling induced by dimethylbenzene in mice (P<0.01)."Puheyin" can significantly reduce toes swelling rate induced by the10%fresh egg white in mice,compared with the model group showed significant difference(P<0.05,P<0.01).The results suggest that anti-inflammatory traditional chinese drug prescription"Puheyin" has significant inhibitory effect on the mice auricle swelling and foot swelling.2Establishment of LPS-induced sepsis in mice and the effect of "Puheyin" on LPS-induced sepsis in miceMouse model by intraperitoneal injection with LPS were used to investigate intervention effects of "Puheyin".In the experiment,there were five groups including control group,model group and drug intervention group.Clinical symptoms of mice were observed at different times. After inoculated with LPS,these mice of model group showed typical clinical symptoms,dysthymic,hair messy,dull and short of breath.The different degree pathological changes were found in the hepar,subcutaneous and intestinal.Histopathological analysis showed that the hepatocyte in mice of model group significant degeneration and swelling,cell borders and nucleus blurred or disappeared nuclei,inflammatory cell infiltration,fatty degeneration of the hepatocyte,apoptotic cells,significantly congestion of hepatic sinusoid,lesions began to gradually reduce after48h,there are many dual-core liver cells at72h.Compared to the model group,symptoms and pathological changes appeared in septic mice that drenched with "Puheyin" were milder,Lobular structure is relatively clear,hepatic cord in hepar arranged mussily,mild degeneration and edema of hepatocytes,had a small amount of inflammatory cell infiltration,little expansion and congestion of central vein.The results showed that traditional Chinese drug prescription "Puheyin" have intervention effects to mouse model of sepsis.3The effects of Puheyin on TLR4and other downstream molecules in the NF-κB signaling pathway in Liver tissue of LPS-induced sepsis in miceInnate immune molecular studies suggested that TLR4,as the main pattern recognition receptors of LPS,causes immune response through TLR4/MyD88/NF-κB signaling pathway.In this experiment,analyzing the change of expression level of important molecules in TLR4/MyD88/NF-κB signaling pathway can preliminary identify anti-inflammatory innate immune mechanisms and target in "Puheyin".In the test,the mRNA expressing level of TLR4,MyD88,NF-κB,TNF-a,IL-10and HMGB1in liver tissue of mice were detected through SYBR Green I qRT-PCR.The results showed that the mRNA expressing level of TLR4,MyD88,NF-κB,TNF-a,IL-10and HMGB1of mice in model group were raising after infected LPS,peaked on18、18、18、12、24、24h,extremely significant difference compared with negative control group(P<0.01).Septic mice were treated by"Puheyin",the mRNA expressing level of TLR4、MyD88、NF-κB、TNF-α、HMGB1of mice were significantly lower than the LPS sepsis model group at12、18、24、24、24h (P<0.05or P<0.01),the mRNA expressing level of IL-10of mice were higher than the LPS sepsis model group,significant difference compared with model group at36h(P<0.05),which suggesting that "Puheyin" may inhibit inflammation through inhibitin the expressing of TLR4、MyD88、NF-κB、TNF-α、HMGB1and promote the expressing of IL-10.Results suggested that TLR4,MyD88,NF-KB,TNF-a,IL-10and HMGB1can be used as the target of "Puheyin"to anti-inflammation in TLR4/MyD88/NF-κB signaling pathways.4The effected of "Puheyin" on TNF-α、IL-10、PGE2、HMGB1in serum of LPS-induced sepsis in miceApart from the examination of mRNA expression above,the double-antibody sandwich ELISA tests were used to detect expression levels of TNF-a,IL-10,PGE2,HMGB1as well as other important related factor protein at different time points in serum of mice.The results showed that the contents of TNF-α、IL-10、PGE2and HMGB1in the serum of mice infected LPS were higher than the control group,and reached the top at12h,24h,18h and24h.In "Model+"Puheyin"group",the content of serum TNF-a was obviously lower than that of the "modle group"at12h,24h(P<0.05),the content of IL-10was outstandingly higher than "modle group" at18h,48h (P<0.05,P<0.01).The content of PGE2was markedly lower than that of "modle group"at18h,24h,36h (P<0.01,P<0.05).The content of HMGB1was obviously lower than that of "modle group" at the time of24h.The results suggested that Puheyin can inhibit the synthesis and releasing of TNF-a,PGE2,HMGB1in serum of sepsis model mice,and promote the synthesis and releasing of IL-10."Puheyin"can regulate synthesis and releasing of important related factor protein as TNF-α、IL-10、PGE2and HMGB1in serum to play a role in anti-inflammtory.The change of important related factor protein in serum is consistent with that of the expression of them in orgnazation.