Studies On Anti-inflammatory And Anti-oxidative Effects Of Liensinine And Its Mechanisms Of Action

Objective:This study was to investigate the anti-oxidative,anti-inflammatory activity and molecular mechanism of Liensinine by establishing the model of LPS on RAW264.7macrophages in cell.On this basis,the anti-inflammatory activity of Liensinine was confirmed by establishing the model of LPS-induced acute lung injury in mice and TPA-induced ear edema in mice in vivo.Methods:we created inflammatory models by LPS-induced in RAW264.7 macrophages in cell experiments.Firstly,CCK8 method was used to screen non-cytotoxic concentration of Liensinine.Flow Cytometry was used to detect the content of ROS in the cells.The content of MDA,the activity of SOD,and the content of GSH in the cells were detected by spectrophotometry.Western blot was used to detect the protein level of Nrf2 / HO-1.The concentration of NO was detected by Griess method.The concentration of PGE2 and the level of pro-inflammatory cytokines were detected by enzyme-linked immunosorbent assay(ELISA).The expression of COX-2 and iNOS mRNA were detected by fluorescent quantitation PCR.Secondly,animal models of acute lung injury and ear edema in mice were performed in vivo,to further confirm the anti-inflammatory activity of Liensinine.BALB/c mice were randomly divided into six group: control group,LPS group,LPS+Liensinine(2 mg/kg,4 mg/kg,8 mg/kg)group,Dexamethasone group.Acute lung injury in mice was induced by nasal instillation of LPS.After 12 h,the pathological changes of lung tissue were observed by histology;The levels of TNF-?,IL-6 and IL-1?in BALF were detected by ELISA;The number of neutrophils in BALF was detected by Wright-Giemsa;Total protein content was detected by BCA method;The pulmonary capillary permeability was detected by Evans Blue;The MPO activity,MDA content,SOD activity,GSH content in lung homogenate supernatant were detected by spectrophotometric;The content of ROS in lung tissue was detected by flow cytometry.The forty ICR mice were randomly divided into Control group,Model group,Liensinine(100 ?g/ear,300 ?g/ear and 600?g/ear)group,Dexamethasone group.In addition to the control group,the other groups were applied TPA to both sides of the right ear simultaneously with Liensinine and Dexamethasone.After 6h,The pathological changes was observed by HE staining.Results:The results of the cell experiments showed that treatment with 0–4(?)?M Liensinine did not show any effect on cell viability,whereas there was an obvious reduction of cell viability at concentration of 8 and 16?M.we used the non-toxic concentrations(0.5,1,2?M)for the subsequent experiment.Therefore,in this study,we used the non-toxic concentrations(0.5,1,2?M)for the subsequent experiment.Liensinine obviously inhibited the production of ROS,decreased the content of MDA,increased the activity of SOD and the content of GSH,reduced the content of NO and PGE2 in inflammatory mediators and reduced the contents of pro-inflammatory cytokine TNF-?,IL-6 and IL-1?in RAW264.7 macrophages induced by LPS;Further protein results showed that the treatment of RAW264.7 macrophages with Liensinine degrade Keap1 protein,release Nrf2 to enter the nucleus,and promote the expression of HO-1 protein,and inhibit I?B?phosphorylation and degradation and NF-?B p65 phosphorylation,suppressed the LPS-induced phosphorylation of p38,JNK and ERK1/2,further down-regulate the mRNA expression of the inflammatory genes iNOS and COX-2.Thus,the anti-oxidative and anti-oxidative activities of Liensinine was elucidated from the molecular mechanism.The results of the cell experiments showed that Liensinine can improve LPS-induced inflammatory cell infiltration in ALI,bronchoalveolar wall thickening andpulmonary congestion,reduce the proinflammatory cytokines TNF-?,IL-6,IL-1? in BALF,decrease the number of neutrophils,the activity of MPO in lung homogenates,the content of protein in BALF,the decrease of pulmonary microvascular permeability,the content of MDA in lung homogenates,the content of ROS in lung tissue,SOD activity and GSH content in lung homogenates,reduce the TPA-induced mouse ear tissue inflammatory cell infiltration and ear sedema,and reduce TPA-induced mouse ear tissue thickening and the ratio of right ear weight / left ear weight.Conclusion:This study confirmed the anti-oxidative effect of Liensinine on LPS-induced RAW264.7 macrophages in molecular mechanism,and further confirmed the anti-inflammatory effect of Liensinine on LPS-induced RAW264.7 macrophages from cellular and molecular mechanisms.In addition,it was confirmed that the safe and effective protective effect of Liensinine on LPS-induced acute lung injury and TPA-induced ear edema inflammatory animal model in mice,providing a theoretical basis for the research on the treatment of inflammatory diseases by Liensinine.