The Protective Function And Mechanism Studies Of Huperzine A On Sepsis In Mice

As a major public health problem,sepsis is one of the most active research areas in modern emergency and critical care medicine.However,its pathogenesis is very complex and has not yet been fully elucidated.Currently,there are no effective drugs available for treating sepsis in clinic.At the early stage of sepsis,there is an imbalance between pro-inflammatory and anti-inflammatory responses,and the synthesis of pro-inflammatory cytokines is dysregulated.This triggers a systemic inflammatory response that ultimately results in death due to multiple organ failure.The systemic regulation of inflammatory response has always permeated the whole pathology of sepsis.Once the homeostasis of immune responses is broken,the body releases a large number of inflammatory factors,resulting in "waterfall release" of inflammatory factors,and the development of sepsis,leading to multiple organ failure.Therefore,the current focus of sepsis treatment is to suppress the inflammatory response and to maintain the dynamic balance of early pro-inflammatory and anti-inflammatory responses.The key is to control"waterfall release" of inflammatory factors at the early stages by various intervention measures.Huperzine A(HupA)is a highly effective acetylcholinesterase inhibitor(ACh EI)and can promote the accumulation of the neurotransmitter acetylcholine(Ach).HupA has shown to enhance learning and memory and improve spatial memory impairment and is used for treating Alzheimer's disease(AD).Some evidence show that HupA has an anti-inflammatory function.However,the mechanism regarding its anti-inflammation activities is unclear.We studied the HupA anti-inflammatory effects against LPS-induced inflammation in cells and mice.The HupA anti-inflammatory effects were futher studied in a mouse sepsis model.We finally explored the mechanism of the HupA anti-inflammatory effects.(1)The LPS-induced inflammatory model was established in RAW264.7 macrophage cell line.RAW 264.7 cells were treated with various concentrations of LPS,and the effects of LPS treatments were evaluated by the CCK-8,real-time fluorescence quantitative PCR and ELISA.The effect of LPS on the expression of nuclear transcription factors NF-?B and AP-1 was determined in RAW-BluetM cells.RAW-BLueTM cells are derived from RAW 264.7 macrophages.They stably express a secreted embryonic alkaline phosphatase(SEAP)geneinducible by NF-?B and AP-1 transcription factors.RAW-BlueTM cells activate NF-kB and/or AP-1 leading to the secretion of SEAP which is easily detectable and measurable when using QUANTI-BlueTM,SEAP detection medium.The results show that the inflammatory response of in vitro cultured RAW264.7 macrophage cells reached the peak 3 h after the treatment with 1.0 mg/L LPS,as indicated by the increase in the levels of the inflammatory cytokines TNF-?,IL-1?,and IL-6(P<0.05).The expression of transcription factors NF-?B and AP-1 in RAW-BlueTM cells were significantly increased(P<0.05),indicating the successful establishment of the LPS-induced inflammation cell model.(2)The LPS-induced inflammation in RAW264.7 macrophage cells were used for the evaluation of HupA anti-inflammatory activity.The results showed that HupA had no significant effect on cell viability at the range of 0-10 ?mol/L.In cells treated with 1.0 mg/L LPS,HupA effectively inhibited the release of inflammatory factors TNF-?,IL-1?,and IL-6 compared to LPS alone(P<0.05),and the nuclear translocation of the transcription factor NF-?B was also effectively inhibited(P<0.05).(3)Based on the above data,we further investigated the effects of HupA on the inflammatory response in the LPS-induced inflammation mouse model.SPF grade C57BL/6 adult mice were randomly divided into 4 groups:untreated control,HupA,LPS model,and LPS+HupA.The optimal drug concentration was determined.The survival rate of mice,body temperature,blood biochemical indicators,serum inflammatory factors,and other parameters were obtained.The histopathological changes were analyzed by hematoxylin and eosin(H&E)staining and immunohistochemistry.The reulsts showed that HupA significantly inhibited LPS-induced inflammation and reduced LPS-induced mortality in mice.The mRNA expression of inflammatory factors IL-6,IL-1?,and TNF-?in the various organs including lung and kidney was significantly attenuated by HupA treatment(P<0.05),with the most notable change in IL-6 mRNA expression.In parallel,the results also showed the similar changes in serum inflammatory cytokine levels as assessed by ELISA.The histopathological analyses found the protective effects of HupA on the organs studied,particularly on lungs.Thus,we conclude that HupA can inhibit the release of inflammatory factors into the serum by regulating their expression in the main organs affected by LPS-induced inflammation in mice.(4)Next,HupA anti-inflammation and protective effects were further assessed in a clinically relevant animal model-cecal ligation and puncture(CLP)sepsis mouse model.After CLP surgery,the survival rate,body temperature,bacteria,routine blood and biochemical indices,cytokines,and histopathology were examined.Significant histopathological damages were found in the organs including the liver and lung in CLP-treated mice and the all other parameters tested were also affected by CLP-treatments.In summary,we successfully establish the CLP mouse model with moderate to severe sepsis presented in typical pathologic tests which was used for further studies in the effects of HupA.(5)The effects of HupA on the CLP-sepsis mouse model with a mortality rate of 80-90%were studied.The mice were randomly divided into 5 groups:sham operation control,CLP alone,CLP with antibiotic(TIENAM),CLP with HupA,and CLPS with HupA+TIENAM group.The survival length and rate of mice were observed,and the protective effects of HupA on these parameters were evaluated.The results show that HupA(0.1 mg/kg d)had an obvious protective effect in CLP mice.The survival rate of CLP-sepsis mice treated with HupA increased to 35%from 15%in untreated CLP mice(P<0.05).Moreover,combining HupA with antibiotics(TIENAM)increased the 7-day survival rate of CLP sepsis mice from the 35%in mice treated with HupA alone to 50%(P<0.05).The expression of inflammatory factors in serum was inhibited to various degrees by HupA treatments,and the pathological damages of sepsis on various organs including the lung and kidney were significantly reduced(P<0.05),indicating that HupA prevented organ failure.Particularly,the NF-?B activity in the lung was significantly inhibited by HupA(P<0.05).The results indicate that HupA can reduce the expression of inflammatory factors in the serum by inhibiting NF-?B activity,preventing tissue damage and organ failure,and improving the survival rate of CLP sepsis mice.(6)Finally,we studied the molecular mechanism of HupA involved in anti-inflammatory and protective effects in CLP-sepsis mouse model.The proteomics studies found a significant difference in the serum expression levels of 308 inflammatory cytokines in the five experimental groups:CLP group,antibiotics(TIENAM)group,HupA group,and HupA+TIENAM group.The data were further studied by Heatmap analysis,PCA analysis,Venn analysis,and Pathway path analysis using KEGG database.The results showed that 18 differentially expressed cytokines were mainly involved in the NF-?B signaling pathway,IGF-1 and IL-10 anti-inflammatory response signal pathways.The NF-?B signal pathway has the highest relevant score.Western blot analysis revealed that HupA inhibited the phosphorylation of I?B? in the NF-?B signaling pathway induced by LPS,thereby reducing the nuclear translocation of the transcription factor NF-?B.HupA also up-regulated the expression of ?7nAChR protein,and the expression level of the phosphorylation of STAT3 and IKBa protein significantly down-regulated.In summary,HupA has a protective effect on sepsis mice,which involves the inhibition on the expression of inflammatory factors.The mechanistic studies reveal that HupA-induced anti-inflammatory functions are associated the regulation of NF-?B signaling,its inhibition of the phosphorylation of IKBa and the subsequent decrease of NF-?B-dependent gene transcription,and it can also activate the ?7nAChR-STAT3 signaling pathway.Future studies are needed to elucidate the signaling pathways downstream of the HupA receptor.This study has revealed the molecular mechanism of HupA-mediated specific anti-inflammatory effect,and laid a solid foundation for the future clincal use of HupA in sepsis.