The Cold-stress Protection Properties Of Selaginella Tamariscina Extract And Amentoflavone And Possible Mechanisms

China has a vast territory with a large temperature difference between north and south.Frostbite is one of the common diseases among the workers in the northeast regions.The non-combat attrition caused by the frostbite has become one of the problems in the Cold War operations,which greatly weakened the combat capability of the army,and it is a problem to be solved urgently.The occurrence of frostbite is closely related to the complement system,as well as the downstream immune and inflammatory pathways.The search for complement inhibitors,especially the classical and the alternative pathway inhibitors of complement C3 protein at the key nodes for the prevention and treatment of frostbite,which has a positive significance.Objective:The purpose of this study is to find potential inhibitors of complement C3 from natural products by using computer-aided drug screening technology,to explore their potential frostbite resistance and to explore their possible mechanism of action,and to provide a theoretical basis for the discovery of lead compounds with anti-frostbite activity.Methods:(1)With complement C3 as the target(PDB ID:2A73),the TCM database was selected from virtual screening by computer-aided drug screening to screen out the traditional Chinese medicine components with potential anti-frostbite activity which is well matched with C3.The selected compounds were classified and summarized to determine the main source of Chinese herbal medicines;(2)To establish a shallow frostbite animal model(Ⅰ-Ⅱ degree),and investigate the index of the degree of frostbite toe swelling,screened the Chinese herbal medicine extract with frostbite protection;(3)Investigate the protective effect and dose-relatedness of Selaginella extract and AF against cold injury by detecting indexes such as degree of toe swelling,Doppler perfusion and blood rheology hemorrheology;(4)Flow cytometry and ELISA were used to investigate the effects of the extract of Selaginella and AF inhibited the activation of the complement system;(5)Tissue sections of the vital organs of the mouse(liver and lung),HE staining and immunohistochemistry staining,Immunofluorescence staining and other experiments to detect the level of complement C3,p-Akt and Cox-2 in the tissue,and the protein expression of BCR/Akt/NF-κB signal pathway was detected by Western Blot and the mechanism of inflammation injury induced by AF frostbite was preliminary explored.Result:(1)10 compounds with good match with complement C3(2A73)were finally screened by molecular docking,through literature search analysis,determine the Chinese herbal medicine source;(2)The results of in vivo activity test showed that the extract of Selaginella had a significant inhibitory activity against foot swelling,which had the value of further research and was preliminarily identified as research object;(3)The extract of Selaginella and AF can significantly reduce the degree of the frostbite-bearing mice toes swelling,increase the local blood perfusion in rats with frostbite and decrease the indexes of blood rheology,such as whole blood viscosity and erythrocyte aggregation index.It can significantly reduce the deposition of complement C3 on the surface of red blood cells caused by cold exposure and significantly increase the level of complement C3 in the serum;(4)AF can improve the growth of frostbite mice,the morphology of liver and lung cells,and can significantly reduce the level of complement C3,inhibit the activation of complement system,reduce the expression of immune response related protein Lyn and inhibit the phosphorylation of Akt,and significantly inhibit the activation of nuclear factor NF-κB,reverse the increased level of inflammatory cytokines TNF-α and COX-2 after frostbite.Conclusion:(1)Computer-aided drug screening contributes to rapidly and accurately screen potential anti-frostbite active components targeting complement C3(2A73);(2)Selaginella extract and AF showed significant activity of anti-frostbite injury,the mechanism may be related to inhibition of C3 activation and the inhibition of complement system activation;(3)AF plays a protective role in the injury of important organs(liver and lung)caused by frostbite,and its mechanism may be related to inhibiting the activation of complement C3 and then negatively regulating the activation of BCR and Akt/NF-κB,thereby inhibiting the inflammatory response.In summary,Selaginella extract and AF initially showed frostbite resistance and could be further developed as a potential drug candidate.Background:Probiotics are commonly used in the prevention of antibiotic-associated diarrhea(AAD).However,the optimum regimen remains controversial.Objective:To compare and rank the relative efficacy and tolerability among all available probiotic agents for AAD through a network meta-analysis.Methods:Eligible studies were identified by searching PubMed,Embase,Medline,Cochrane library and Web of Science for randomized controlled trials(RCTs)that examined the efficacy of probiotic therapy for AAD.A random effects model was applied within a frequentist framework.Quality of evidence was performed by GRADE approach.The project was prospectively registered with PROSPERO(CRD 42016050776).Results:Fifty-one articles(60 comparisons,9569 participants),including ten probiotic interventions,were identified.LGG(Lactobacillus rhamnosus GG)had the highest probability of being ranked best in both effectiveness(odds ratio[OR],95%confidence interval[CI]=0.28[0.17,0.47])and tolerance(0.44[0.23,0.84])on prevention of AAD.With regard to reducing Clostridium difficile(C.difficile)infection rate,L.casei(Lactobacillus casei)was considered better efficacy(0.04[0.00.0.77])and medium tolerance(0.56[0.19,1.66]).Strain combination reported no superiority over single strain in either efficacy or tolerability.Conclusions:LGG is probably the best option to consider when AAD is indicated.L.casei appears to be the most efficacious choice when associated with severe C.difficile-related cases.