Anti-inflammatory Pain Effect And Mechanism Of Alcohol Extract From Tujia Medicine KHS

Objective:The KHS is a drug commonly used by the Tujia family for hurricane dehumidification,gas and pain relief in Tujia.It has been identified as the cane of Kadsura heteroclita（Roxb）Craib,a genus of Schisandra chinensis.It is used by folks to treat rheumatic pain,bone pain and rheumatoid arthritis.A model of inflammatory pain in formalin-induced pain mice,heat-stimulated mice and glacial acetic acid-induced pain mice were established to research the pharmacodynamics and related mechanisms of anti-inflammatory pain of KHS.In addition,inflammation model of mouse monocyte macrophage RAW264.7 in vitro was stimulated by Lipopolysaccharide（LPS）in order to explore its anti-inflammatory mechanism.Therefore,the mechanism of anti-inflammatory pain of Tujia medicine KHS is clarified,and providing a basis for researching and developing it as a safe and effective national innovative medicine.Methods:1.The analgesic effects of KHS alcohol extracts were evaluated by establishing formalin-induced pain mice,heat-stimulated mice,and glacial acetic acid-induced pain models.2.By replicating formalin-induced inflammatory pain model in mice and detecting the content of PGE₂,TNF-α,IL-1βand IL-6 inflammatory factors in the swollen inflammation tissue of the model mice using ELISA kit,mechanism of analgesic of KHS alcohol extracts was investigated.3.By replicating the inflammatory pain model induced by glacial acetic acid in mice and detecting the content of TNF-αand IL-6 inflammatory factors the in the serum of the model mice using ELISA kit,mechanism of analgesic of KHS alcohol extracts was explored.4.In order to explore the anti-inflammatory mechanism of KHS alcohol extracts,inflammation model of mouse mononuclear macrophage RAW264.7 was stimulated by lipopolysaccharide（LPS）in vitro,and the cytotoxicity of the drug was determined by CCK-8 method and the appropriate experimental concentration of the drug was screened,as well as,The effect of KHS alcohol extracts on the expression of macrophage inflammatory cytokines（TNF-α,IL-6 and NO）stimulated by LPS were detected by ELISA kit and the Griess method.Results:1.Compared with the model group,the KHS alcohol extract group inhibited the cumulative lameness time of the first phase,the second phase and the total pain reduction score of the formalin-induced inflammatory pain mice（P<0.05 or P<0.01）.The inhibition rate copepod response in the first phase in formalin-induced inflammatory pain mice was 14.70%,16.33%and 19.41%in KHS alcohol extract group with different dosage respectively,and it in the second phase phase were48.69%,39.04%and 53.47%,respectively.2.Compared with the model group,the KHS alcohol extracts group with different dose(200,400,800 mg·kg^-1)prolonged the latency of the foot in the heat-stimulated mice and increased the pain threshold.What’s more,the analgesic effect of the KHS ethanol extracts was significant in each dose group at 60 min and 90 min after administration（P<0.01）.3.Compared with the model group,the KHS alcohol extracts(200,400,800 mg·kg^-1)prolonged the writhing latency and inhibited the number of writhing in glacial acetic acid-induced mice in a dose-dependent manner.The inhibitory rates of writhing reaction were 55.17%,57.57%and 71.60%,respectively.4.Compared with the model group,the KHS alcohol extracts in each dose group(200,400 and 800 mg·kg^-1)dose-dependently inhibited the expression of PGE₂,TNF-α,IL-6 and IL-1βin the inflammatory inflamed tissue of mice with formalin-induced inflammatory pain（P<0.05,P<0.01 or P<0.001）.5.Compared with the model group,the KHS alcohol extracts(200,400 and 800mg·kg^-1)dose-dependently inhibited the expression of TNF-αand IL-6 in the serum of glacial acetic acid-induced inflammatory pain mice（P<0.05,P<0.01 or P<0.001）.6.The results of CCK-8 experiment showed that KHS alcohol extracts(125～1000μg·m L^-1)had more significant effect in 24 hours than in 48 hours.The experimental results of LPS-induced inflammation in RAW264.7 cells showed that KHS alcohol extracts began to inhibit the cell viability from 200μg·m L^-1,but there was no statistical difference;the KHS alcohol extracts 800μg·m L^-1 combined with100ng·m L^-1 LPS showed obvious inhibition of cell viability.7.The ethanol extract of KHS inhibited the expression of inflammatory factors TNF-α,IL-6 and NO in the RAW264.7 cell inflammation model induced by LPS in a dose-dependent manner.Conclusions:The alcohol extract of Tujia medicine KHS had a good anti-inflammatory and analgesic effects,and its mechanism may be related to the the production of inflammatory cytokines PGE₂、TNF-α,IL-1βand IL-6 and the inhibition of NO expression.