Protective Effects And Mechanisms Of Penehyclidine Hydrochloride On Acute Lung Injury Induced By Double-hit In Rats

Part One:Role of TLR4/NF-?B signal pathway in the double-hits induced acute lung injury in ratsObjective:To establish the animal model of double-hit acute lung injury (ALI) induced by blunt chest trauma and hemorrhagic shock and explore the effects of TLR4/NF-?B signal pathway in rat models of ALI.Methods:Forty male Sprague-Dawley (SD) rats weighing240to280g were randomly assigned into four equal groups (n=10):Sham (Sham) group, blunt chest trauma (T) group, hemorrhagic shock (HS) group, blunt chest trauma and hemorrhagic shock (THS) group. Sham group only underwent femoral arterial and venous puncture without blunt chest trauma and hemorrhage; T group establish blunt chest trauma model; HS group establish hemorrhagic shock model and the THS group establish the animal model of double-hit acute lung injury. The heart rate (HR), respiratory rate (RR) and mean arterial pressure (MAP) changes of the rats were observed. The arterial blood samples were collected for gas analysis at6hours after infection. The expression of lung TLR4and NF-?B detected with the method of Western blotting and immunochistochemistry. The serum tumor necrosis factor-a (TNF-a), interleukin-6(IL-6) and interleukin-1?(IL-1?)levels were measured using enzyme-linked immunosorbent assays (ELISA). Light and electron microscopy were also performed to detect the injury of lung tissue.Results:T, HS, THS group had significantly higher HR and R than corresponding Sham group (P<0.05); MAP and PaO2was significantly decreased in T, HS, THS group versus C group (P<0.05).Significantly increased expressions of TLR4and NF-?B protein in lung tissue were observed in group THS and the serum contents of TNF-a, IL-6and IL-1?in group THS were higher than those in group T and group HS (P<0.05) and also significantly higher than those in group Sham. The lung histopathologic damage was significantly aggravated in THS group as compared with other groups.Conclusions:TLR4/NF-?B is the pivotal signal pathway in double-hit induced ALI, and play an promotion role on the delayed-development of it. Part Two:Protective effects of penehyclidine hydrochloride on acute lung injury induced by double-hit in ratsObjective:To observe the effect of penehyclidine hydrochloride (PHC)on the arterial blood gas, lung permeability index (LPI), the protein content of bronchoalveolar lavage fluid (BALF) and polymorphonuclear neutrophils (PMN) percentage, myeloperoxidase (MPO) activity, lung water content (LC), lung wet/dry weight (W/D), and explore its protective effect on double-hit induced by ALI in rats.Methods:Forty male Sprague-Dawley (SD) rats weighing250to300g were randomly assigned into four equal groups (n=10):Sham (Sham) group, double-hit ALI (THS) group, PHC for prevention (P1) group, PHC for treatment (P2) group. Rat models of double-hit ALI were used in this study. Blood and lung tissue samples were collected after6h. LPI, protein content and PMN percentage in bronchoalveolar lavage fluid (BALF), MPO activity, lung water content (LC), W/D ratio and arterial blood gas analysis were measured. The pulmonary pathologic changes were observed under light microscopy and electron microscopy.Results:Double hit-challenged rat had significantly increased LPI, protein content in BALF, PMN percentage, MPO activity, lung water content,W/D ratio, PaCO2, Lac (P<0.01) and decreased PaO2, pH (P<0.01). Administration of PHC resulted in a significant increase PaO2, pH (P<0.01or P<0.05) and decreased LPI, protein content in BALF, MPO activity, PMN percentage, lung water content, W/D ratio PaCO2and Lac (P<0.01or P<0.05). PHC treatment resulted in a marked attenuation of those pathologic alterations.Conclusions:These results demonstrate that prevention and treatment PHC can attenuate ALI and exert protective effect on ALI induced by double-hit Part Three:Effects of penehyclidine hydrochloride onTLR4/p38MAPK signal transduction pathway in actue lung injury induced by double-hit in ratsObjective:To establish the animal model of double-hit acute lung injury (ALI) induced by blunt chest trauma and hemorrhagic shock, and investigate the effects of hydrochloric penehyclidine (PHC) on TLR4/p38MAPK signal transduction pathway in actue lung injury induced by double-hit in rats.Methods:Forty male Sprague-Dawley (SD) rats weighing250to300g were randomly assigned into four equal groups (n=10):Sham (Sham) group, double-hit ALI (THS) group, PHC for prevention (P1) group, PHC for treatment (P2) group. Rat models of double-hit ALI were used in this study. The blood and lung samples were collected at6hours after infection. Protein levels of TLR4and p38MAPK in lung were assessed by western-blot and immunofluorescence assay. NF-?B and AP-1activation was measured with electrophoretic mobility shift assay (EMSA). The concentrations of necrosis factor-a (TNF-a), interleukin-6(IL-6) and interleukin-1?(IL-1?) in the serum were measured by enzyme-linked immunosorbent assays (ELISA).Results:Compared with sham group, TLR4and p38MAPK protein expression, NF-?B and AP-1activation, TNF-a?IL-6andIL-1? level were markedly increased in THS group (P<0.01). Compared with THS group, TLR4and p38MAPK protein expression, NF-?B and AP-1activation, TNF-??IL-6and IL-1?level were obviously lower in P1group and P2group (P<0.01)Conclusions:The alteration of TLR4/p38MAPK dependent signal pathway in lungs is closely connected with double-hit induced ALI. PHC can effectively control the elevation of TLR4/p38MAPK expressions in lung and can inhibit the release of inflammatory mediators. TLR4/p38MAPK signal pathway is closely related to double-hit induced by ALI.These results suggest that PHC attenuates double-hit induced acute lung responses through inhibition of TLR4and p38MAPK expressions, reduce NF-?B and AP-1DNA binding activity and reduce the release of inflammatory cytokines to curb the inflammatory cascade, further reduce tissue inflammatory injury.