| Background: The inflammatory response is present throughout human life and is a common feature in almost all pathological conditions.As a key part of the immune activity process of the organism,the inflammatory response in a broad sense can be understood as the process of removing foreign pathogens or internal necrotic cells by the organism in the face of infection;in a narrow sense,the inflammatory response involves the immune response of cells deployed by a storm of factors generated by each tissue cell in the face of external stimuli.Ultimately,the starting point of the inflammatory response is to protect the organism from damage.However,the intricate response,when overdone,can cause more serious damage to the body and worsen the disease.In the face of such thorny issues of inflammatory responses,there has been a clinical expectation for safer and lower risk natural drugs to emerge and to treat inflammation efficiently.Research related to the anti-inflammatory effects of traditional Chinese medicine compounds has become an important research area in recent years,and clinical studies on the anti-inflammatory effects of Chinese medicinal preparations have made some progress,but the specific active ingredients and mechanisms of action still need to be further clarified.Solanum Lyratum Thunb is a commonly used traditional Chinese medicine with purgative and detoxifying effects,which is currently biased toward the study of its anti-cancer effects.In particular,the steroid alkaloid(SA)component in Solanum Lyratum Thunb has been increasingly reported to explore its biological activity,suggesting that Solanum Lyratum Thunb SA has good research prospects.In this study,we selected steroid alkaloid compounds extracted from Solanum Lyratum Thunb as the test drug and used TLR4 protein as the main entry point to investigate the anti-inflammatory effects of this compound and the anti-inflammatory mechanism associated with TLR4/My D88/NF-κB signaling pathway.This paper is expected to meet the urgent need for clinical anti-inflammatory drug use and provide new ideas for the treatment of inflammatory response,as well as new evidence for the development of using Solanum Lyratum Thunb as a candidate anti-inflammatory Chinese medicine.Methods: For animal experiments,we investigated the effects of Solanum Lyratum Thunb SA on acute inflammation in mice by constructing an inflammatory model of ear swelling caused by xylene and detecting the weight,and histological changes of mouse ear pieces;we constructed a model of acute abdominal inflammation induced by thioglycolate broth in mice and counted the changes of mouse abdominal leukocyte concentration using cell counting plates;we detected the pro-inflammatory factors IL-1β,IL-6 and TNF-α in mouse serum by ELISA kits.IL-6 and TNF-α were measured by ELISA kit.The experiment was divided into blank group,model group,positive control group and Solanum Lyratum Thunb SA low(0.25mg/kg),medium(1mg/kg)and high(2mg/kg)concentration drug groups.The administration method was tail vein injection administration.For cellular experiments,RAW264.7 cell inflammation model was constructed using Lipopolysaccharide(LPS),and the effects of different concentrations of Solanum Lyratum Thunb SA on cell viability were detected by CCK8 assay;ELISA and RT-q PCR assays were performed to detect the effects of different concentrations of Solanum Lyratum Thunb SA on macrophage polarization characteristic factors IL-12,IL-10 and pro-inflammatory factors IL-1β and IL-1β.The expression changes of IL-1β,IL-6 and TNF-α were detected by ELISA and RT-q PCR.The experiments were divided into blank group,model group,positive control group and Solanum Lyratum Thunb SA low(5μg/ml),medium(20μg/ml)and high(40μg/ml)concentration drug groups.For molecular experiments,the inflammation model of RAW264.7 cells was constructed using LPS,and the effects of different concentrations of Solanum Lyratum Thunb SA on the expression of TLR4 protein on the cell surface were detected by flow cytometry,cellular immunofluorescence and western blot techniques were used to detect changes in the expression of total TLR4 protein in cells,finally,western blot experiments were used to explore the effects of Solanum Lyratum Thunb SA on TLR4/My D88/NF-κB signaling pathway related protein expression levels.The experiments were divided into blank group,model group,positive control group and Solanum Lyratum Thunb SA low(5μg/ml),medium(20μg/ml)and high(40μg/ml)concentration drug groups.Results: Both the medium and high concentration groups of Solanum Lyratum Thunb SA could significantly inhibit ear swelling and improve the symptoms of transverse muscle rupture,vasodilation and congestion caused by xylene in mouse ear tissues;all concentrations of Solanum Lyratum Thunb SA could reduce the number of mouse abdominal leukocytes and significantly inhibit the expression of pro-inflammatory factors IL-1β,IL-6 and TNF-α in mouse serum in a concentration-dependent manner,especially the effect of medium and high concentrations of Solanum Lyratum Thunb SA was more significant.On the basis of ensuring that the concentration of Solanum Lyratum Thunb SA did not affect the cell viability of RAW264.7 and had no obvious toxic effect,it was found that Solanum Lyratum Thunb SA could inhibit IL-12 molecules and promote the expression and secretion of IL-10 molecules in the M1 polarized state of macrophages,and the higher the concentration,the more significant the effect was.In addition,Solanum Lyratum Thunb SA could effectively inhibit the expression of pro-inflammatory factors IL-1β,IL-6 and TNF-α in RAW264.7 cells;inhibit the expression of TLR4 protein on cell membrane,and inhibit the expression of each protein of TLR4/My D88/NF-κB signaling pathway.Conclusion: Solanum Lyratum Thunb SA has significant anti-inflammatory effects in a concentration-dependent manner,Solanum Lyratum Thunb SA can mediate anti-inflammatory effects through polarizing macrophage RAW264.7 and through inhibiting TLR4/My D88/NF-κB cascade reaction. |
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