Role And Mechanism Of ROS On Post-hemorrhagic Shock Mesenteric Lymph Mediating Acuted Lung Injury

Acute lung injury(ALI)is the result of pathogenic factors leading to severe inflammatory response,pulmonary vascular hyper-permeability,and acute pulmonary edema.Due to the pulmonary hemodynamic characteristics and hemorrhagic shock-induced microcirculation dysfunction,ALI is the most likely to occur in patients with hemorrhagic shock,and easily develop to acute respiratory distress syndrome(ARDS),which can endanger life.Previous studies in our lab showed that the reduction of mesenteric lymph return alleviated the lung injury,and reduced the expressions of toll-like receptor 2(TLR2),TLR4,and high-mobility group box1(HMGB1)in lung tissue.Some studies showed that reactive oxygen species(ROS)could lead to ALI through stimulating the body's inflammatory response,mediating apoptosis and increasing vascular permeability following hemorrhagic shock.Howver,it is remain unclear that whether ROS involves in the ALI mediated by post-hemorrhagic shock mesenteric lymph(PHSML),whether the reduction of ROS generation and releasing is related to decreasing the return,and whether generation and releasing of ROS is affected by TLR2,TRL4 and HMGB1,which are need further investigated.Therefore,based on the previous studies,we observed the effects of drainage of PHSML on the organization structure of pulmonary tissue and the ROS content following hemorrhagic shock,intravenous infusion of PHSML on the pulmonary histology,ROS content,the expressions of TLR2,TLR4,HMGB1 in the mice(Wild type,TLR2-/-,TLR4-/-).Furthermore,we observed the effect of PHSML on the generation andreleasing of ROS in pulmonary microvascular endothelial cells(PMVECs),and the effects of the ROS inhibitor,blocking antibody of TLR2 and TLR4 on PHSML,so as to determine the PHSML is one of the factor that can increase the generation and releasing of ROS,and its mechanism is mediated by TLR2,TLR4 and HMGB1.First of all,eighteen C57BL/6J wild type male mice were randomly divided into three groups: sham group,hemorrhagic shock group(shock),shock + drainage group,for the observation of the effect of PHSML on the structure of lung tissue and ROS content by using hemorrhagic shock model.Results showed that the structure of lung tissue was normal in sham group;The structure of alveoli was severely disrupted and coalesced,the alveolar wall tissue was thickened,and a large amount of inflammatory cells were infiltrated in shock group;The degree of lung injury was milder in the shock+ drainage group than that of the shock group.Meanwhile,the content of ROS was increased significantly in lung tissue of mice following hemorrhagic shock,which was ROS reduced significantly by PHAML drainage.Then TLR2-/-and TLR4-/-mice were respectively divided into the sham and shock groups,and the effect of TLR2,TLR4 knock out on the organization structure of lung tissue and ROS content were observed.Results showed that there were inflammatory material in alveolar interval,and the partial structure of alveoli was disrupted and coalesced in shock group of TLR2-/-/TLR4-/-;but the degree was wilder compared with the WT mice in the shock group,and TLR2 and TLR4 deficiency alleviate hemorrhagic shock-increased ROS in murine pulmonary tissues in WT mice.At last,collect normal mesenteric lymph(NML)and PHSML by using fifty WT mice,and respectively transfuse to WT,TLR2-/-,TLR4-/-mice,six mice respectively,and observed the effect of transfusion with PHSML on the organization structure of lung tissue,ROS content and theexpression of TLR2,TLR4 mRNA.Results showed that the intravenous injection of NML has no significant effect on TLR2-/-,TLR4-/-and WT mice;while the intravenous injection of PHSML induced lung injury of WT mice was more severe compared with TLR2-/-,TLR4-/-.Meanwhile the intravenous injection of PHSML increased ROS content and the expression of mRNA,and both significantly higher than the intravenous injection of NML in WT mice.The intravenous injection of PHSML increased ROS in lung tissue TLR2-/-,TLR4-/-mice were significantly lower than WT mice and higher than the homotypic mice.The intravenous injection of PHSML increased the expression of TLR2 mRNA in TLR4-/-mice,and significantly higher than the intravenous injection of NML and WT mice injected PHSML;Also,the intravenous injection of PHSML increased the expression of TLR4 mRNA in TLR2-/-mice,and significantly higher than the intravenous injection of NML and WT mice injected PHSML.In order to investigate the role of ROS on ALI mediated by PHSML,and the relation with TLR2,TLR4,we take PMVECs as target,observed the effect of PHSML contribute to increase the generation and releasing of ROS by using the inhibitor(NAC),TLR2 and TLR4 inhibitors blocking antibody.Results showed that the PHSML caused the injury of PMVECs and the production of ROS was increased.NAC,TLR2 and TLR4 blocking antibody both alleviated the injury of the structure of PMVECs,and inhibited the production of ROS in PMVECs mediated by PHSML.In addition,in order to investigate the role of HMGB1 on ALI mediated by PHSML,we observed the structure of lung tissue and the content of ROS in the normal mice through the intravenous injection of PHSML after the intraperitoneal injection of glycyrrhizic acid(GL),an inhibitor of HMGB1.Results showed that pretreatment with GL significantly reduced the lung injury and the production of ROS by intravenous infusion of PHSML in normal mice.These results suggest thatHMGB1 involved in the mechanism of ALI result from ROS mediated PHSML.This study showed that PHSML cause excessive generation of ROS and mediate lung tissue injury is one of the important mechanisms in ALI following hemorrhagic shock;PHSML mediated lung injury and the mechanism of excessive generation and releasing of ROS following ALI mediated by PHSML is related to TLR2 and TLR4,HMGB1.Take PHSML,ROS as target,can contribute to prevent and treatment of ALI caused by severe shock.