Shenfu Injection In The Treatment Of Endotoxin Shock By Regulating The Combination Of HMGB1 And NF-KB

ObjectiveEndotoxin shock belongs to the category of syncope disease in Traditional Chinese Medicine(TCM),and its pathogenesis is closely related to the cascade effect of inflammatory factors.The study about the molecular mechanism of inflammatory reaction is mainly focused on NF-κB P65(P65),which is key for the transcription of inflammatory factors.High mobility group box-1 protein(HMGB1)is a highly conserved nucleoprotein,Extracellular HMGB1 can activate the MAPKs and NF-κB signaling pathways and then promote the release of a variety of inflammatory cytokines as well as HMGB1.Therefore,extracellular HMGB1 is considered to be the key factor of the lethality of late endotoxin shock,and the inhibition of its expression and secretion may be an important therapy for endotoxin shock.Shen Fu injection derived from Si Ni Jia Ren Shen Decoction in "Treatise on Febrile Disease".In this formula,red ginseng is the sovereign drug,it can tonify qi and prevent exhaustion;monkshood is the ministerial drug,it can invigorate yang in heart and kidney.Taken together,this formula can rescue from collapse by restoring yang,nourish qi to stop collapse.Because of its obvious curative efect,this formula is often utilized in clinic for the syndrome of exhaustion of Yang such as infectious shock or hemorrhagic shock.Since the mechanism of the curative effect is still unclear,more research is required.Our previous study found that Shen Fu injection could inhibit the translocation of HMGB1 from nucleus to cytoplasm and the activation of NF-κB.Some reports also found that HMGB1 has a bond with the Rel family members.According to our previous studies,we hypothesized that the Shen Fu injection may inhibit the transcription of inflammatory factors by regulating the binding between HMGB1 and members of the NF-κB family.In this study,we explore the molecular mechanism about how Shen Fu injection regulate HMGB1 and treat endotoxin shock by establishing the cell and animal model of endotoxin shock.Methods1.SD rats were selected to establish the animal model of endotoxin shock,Shen Fu injection at low,medium,high dose and the positive control drug(dexamethasone)was injected as treatment.Physio graph was used to monitor the average arterial pressure(MAP)in rats for 6 hours,the mortality rate within 72 hours was also observed,thus determine the effect of S hen Fu injection on average arterial pressure and mortality in rats with endotoxin shock.Lung tissue was taken from the rats for the subsequent test.The protective effect of Shen Fu injection on acute lung injury was observed by HE staining.RT-PCR was used to observe the influence of Shen Fu injection on the transcription of the proteins(HMGB1,P65 and P50)related to HMGB1/NF-κB signaling pathway in lung tissue and their downstream inflammatory factors.Western blot was used to detect the total,nuclear and cytoplasm expression quantity of the proteins related to HMGB1-NF-κB signaling pathway in lung tissue to determine the influence of Shen Fu injection on the nuclear transposition of HMGB1 and P65.Elisa was used to detect the influence of Shen Fu injection on the level of inflammatory factors in rat plasma.2.RAW264.7 cells were stimulated by LPS to establish the cell model of inflammation,and then be treated by S hen Fu injection at low,medium,high dose and the positive control drug(histone acetylation enzyme inhibitors RGFP966)respectively.Immunofluorescence staining was used to observe the influence of Shen Fu injection on HMGB1 and P65 nuclear translocation qRT-PCR was used to observe the influence of Shen Fu injection on the transcription of the proteins(HMGB1 P65 and P50)related to HMGB1/NF-κB signaling pathway and their downstream inflammatory factor TNFα.Western blot was used to observe the total,nuclear and cytoplasm expression quantity of the proteins related to HMGB1-NF-κB signaling pathway to determine the influence of S hen Fu injection on the nuclear transposition of HMGB1 and P65.Elisa was used to detect the influence of S hen Fu injection on the secretion level of inflammatory factors in cellular supernatant.3.Mammalian Two-Hybrid system was used,the CDS areas(mice original)of HMGB1,P65 and P50 genes were cloned into the pACT and pBIND carriers respectively,trans fect 293t cell transiently with positive control plasmid(pACT-M yod and pBIND-Id)as reference.The inflammation model was established by TNFα stimulation,and Shen Fu injection was given as treatment.Dual Luciferase Reporter was used to detect the influence of Shen Fu injection on the combination and binding force of each two among the three proteins(HMGB1,P65 and P50).4.RAW264.7 cells were stimulated by LPS to establish the cell model of inflammation,and then be treated by Shen Fu injection at low,medium,high dose and the positive control drug(RGFP966)respectively.Co-IP was used to detect the influence of Shen Fu injection on the combination and binding force of each two among the three proteins(HMGB1,P65 and P50)within cells.5.Tet-on-HMGB1+-RAW264.7 cells were constructed to observe the transcription,exression and nuclear translocation of HMGB1 and P65 after stimulating with LPS,and to explore the role of HMGB1 in the process of inflammation.6.Combination of HMGB1 and P65 as well as HMGB1 and P50 in Tet-on-HMGB1+-RAW264.7 cells were detected by Co-IP method,and compared with the combination that in wild type RAW264.7 cells,and try to explor the possible molecular mechanism of Shenfu Injection in treatment of endotoxic shock HMGB1 regulationResults1.The mean arterial pressure was significantly reduced in rats with endotoxin shock,and the mortality rate within 72 hours was 64.3%.The blood pressure of shock rats could be improved by Shen Fu injection of each dose.The mortality of rats could be significantly reduced by Shen Fu injection,the medium dose group decreased to 28.6%,which was better than other treatment groups.2.The lung tissue of endotoxin shock rats was shown as typical acute lung injury with pulmonary alveolar edema,hyperemia,and pulmonary inflammatory cell infiltrationShen Fu injection could alleviate the lung tissue damage and pulmonary inflammatory response induced by LPS.Among all the groups,the effect of the medium dose group wasthe best,better than low,high dose and dexamethasone group.3.The transcription and expression levels of HMGB1/NF-κB signaling pathway related proteins(HMGB1,p65,p50)were significantly increased in the lung tissues of endotoxin shock rats.Shen Fu injection could inhibit the activation of HMGB1-NF-κB signaling pathway.4.HMGB1 and TNF a were significantly increased in the plasma of endotoxin shock rats,while the growth factor(TGF)and IL-10 were significantly decreased.Shen Fu injection could inhibit the release of proinflammatory cytokines and increase the secretion of anti-inflammatory factors.5.LPS induced the transcription,expression,extranuclear migration and extracellular release of HMGB1 in RAW264.7 cell.Shen Fu injection could inhibit the transcription,expression,extranuclear migration and release of HMGB1 effectively.6.LPS induced the expression of P65 in RAW264.7 cells,and P65 was partially transferred from cytoplasm to the nucleus,Shen Fu injection could inhibit the expression and nuclear translocation of NF-kappa B P65.7.LPS induced a significant increase of HMGB1,TNFα,interleukin-1(IL-1)and induced nitric oxide synthase(iNos)in RAW264.7 cells,Shen Fu injection could inhibit the release of the above proinflammatory factors.8.The results of Mammalian Two-Hybrid system suggested that there existed combinations between HMGB1 and P65,HMGB1 and P50,P65 and P50.Shen Fu injection could increase the combination of HMGB1 and P65,HMGB1 and P50,and reduce the combination of P65 and P50.9.Results of Co-IP showed that HMGB1 within cells could be combined with-P65 and P50 respectively.LPS could significantly reduce the binding rate between HMGB1 and P65,HMGB1 and P50,while each dose of Shen Fu injection could significantly increase the binding rate between HMGB1 and P65.10.The tolerance of Tet-on-HMGB1+-RAW264.7 cells to LPS was significantly stronger than that of wild type cells(0.2 u g/mL).The dosage of LPS needed to be increased to 1 u g/mL to induce the translocation of HMGB1.11.Shenfu injection can inhibit the expression and nuclear transposition of HMGB1 and P65 in LPS-induced Tet-on-HMGB1+-RAW264.7 cells.12.The overexpression of HMGB1 in the nucleus increased the combination of HMGB1 and P50,especiafly in the high dose group of Shenfu injection.Conclusion:1.Shen Fu injection could inhibit LPS-induced shock and lung injury by inhibiting the proteins related to HMGB1一NF-κB signaling pathway.2.Shen Fu injection could alleviate the inflammatory injury by inhibiting the secretion of pro-inflammatory factors and promoting the release of anti-inflammatory factors.3.The combination of HMGB1 and P65,HMGB1 and P50 can be enhanced by Shen Fu injection,while inhibiting the combination of P65 and P50,which may be one of the important mechanisms for the treatment of inflammation and shock.4.The overexpression of HMGB1 in the nucleus increased the tolerance of the cells to LPS,and could change the binding ratio of HMGB1 to P65 as well as HMGB1 to P50.5.Shenfu injection can regulate the combined with HMGB1 to P65 as well as HMGB1 to P50,thus inhibite the inflammatory reaction,which may be an important mechanism of the treatment of shenfu injetion with endotoxic shock.