Study On The Molecular Mechanism Of Gentiopicroside By Regulating The ASK1-mediated P38 MAPK Signaling Pathway To Alleviate Pulmonary Inflammatory Damage Caused By Paragonimus Proliferus

Objective:To establish a pulmonary injury model in rats infected by Paragonimus proliferus,to investigate whether Gentiopicroside can regulate the ASK1-mediated p38 MAPK signaling pathway to alleviate pulmonary inflammatory damage caused by Paragonimus proliferus.Methods:63 SD male rats(6-8weeks/180-200g)were obtained from experimental animal department of Kunming Medical University.In addition to the 10 rats in the normal group,the other 53 rats were injected into the abdominal wall of each rat to receive 6 metacercariae subcutaneously.After 6 weeks of infection,3 rats were randomly selected for dissection and morphological observation to make sure whether the model was successful.After the models established successfully,the remaining 50 rats were given insecticidal treatment by oral administration with 200 mg/kg/d trichlorobendazole,one time a day.After 4 days of continuous gavage,50 rats were randomly average divided into 5 groups(Control/Model/LD Gent/MD Gent/HD Gent/Colchicine)with dates and marks.10 rats were given to the model group with 0.2ml of normal saline.Gentiopicroside treatment groups orally administered at a low dose of 50 mg/kg/d,a medium dose of 100 mg/kg/d,and a high dose of 200 mg/kg/d,10 rats for each dose group.The positive drug control group was given colchicine 0.25mg/kg/day.The rats in each of the above five groups were treated by continuous gavage,once a day,for 4 weeks.Anesthetized on an empty stomach,rat serum was used for ELISA experiments to determine the expression of inflammatory cytokines IL-1 and IL-6.The lung tissues were subjected to HE staining,Western Blot and IHC experiments to observe the morphological changes and molecular detection.The expression of ASK1,MKK3,p-MKK3,p38,p-p38,MSK1 and NF-κB p65 were detected in the ASK1-mediated p38 MAPK signal pathway.Results:Lung tissues were observed by HE staining,lung vacuole structure of rats infected by Paragonimus proliferus was destroyed in the model group,under the treatment of Gentiopicroside,vacuole structure of lung was gradually restored compared with the model group;In the ELISA experiment,the expression of inflammatory cytokines IL-1 and IL-6 in rat serum infected by Paragonimus proliferus were higher than those in the control group,and inflammatory cytokines IL-1 and IL-6 in the treatment groups of Gentiopicroside were decreased with different degrees,among which the high-dose group had the best therapeutic effect;In the Western Blot and IHC experiments,compared with the control group,the expression of ASK1,MSK1,NF-κB p65 were increased obviously,but the changes of MKK3 and p38 were not evident,the level of protein phosphorylation(p-MKK3ser189,p-p38Thr180/Tyr182)have significantly raised.ASK1,MKK3,p-MKK3,p38,p-p38,MSK1 and NF-κB p65 were significantly down-regulated after treatment by Gentiopicroside compared with the model group.Conclusion:In the rat lung injury model caused by Paragonimus proliferus infection,the activation of p38 MAPK signaling pathway by ASK1 caused pulmonary inflammatory response.Gentiopicroside can regulate the the ASK 1-mediated p38 MAPK signaling pathway to relieve pulmonary inflammatory damage in rats induced by Paragonimus proliferus infection,and there is a certain dose dependence.