| Objective:To explore the effects and mechanisms of tetramethylpyrazine(TMP)on acute lung injury induced by endotoxin(LPS).Methods: 1)To establish the model of " double hit" by LPS on rat,20 healthy adult SD rats were randomly divided into 4 groups:(1)control group: injection of an equal saline instead of LPS,(2)LPS group: intraperitoneal injection of LPS(1mg/kg),16 hours later,rats were treated by intratracheal instillation of LPS(3 mg/kg),and the ALI rat models were established.(3)TMP1 group: After establishing the model of ALI,rats were treated with TMP(40 mg/kg)for the prevention and treatment,(4)TMP2 group: After establishing the model of ALI,rats were treated withTMP(80mg/kg)for the prevention and treatment.2)To detect lung wet/dry weight ratio.3)Lung histopathological examination by HE staining.4)The expression of ROCK2,LIMK1 and cofilin in lung tissue were detected by Western blot.5)HUVECs were randomly divided into six groups:(1)control group(ECM complete medium)(2)LPS group(LPS 0.1ug/ml)(3)TMP group(TMP 1.5mg/ml)(4)TMP1 group(TMP 0.5 mg/ml+LPS 0.1 ?g/ml)(5)TMP2 group(TMP 1 mg/ml+LPS 0.1 ?g/ml)(6)TMP3 group(TMP 1.5 mg/ml+ LPS 0.1ug/ml).Pretreatment: The control and LPS group were added with the equal amount of ECM complete medium,and the remaining four groups were respectively added with the corresponding concentrations of tetramethylpyrazine for 12 hours.After the pretreatment,equal amounts of medium were added to the control and TMP group,and the remaining four groups were cultured with LPS for 12 hours.6)Using immunofluorescence method to observe the changes of the morphology and distribution of endothelial cytoskeleton.7)The expression of ROCK2,LIMK1,p-LIMK1,cofilin and p-cofilin in endothelial cells were detected by Western blot.Results: 1)wet/dry weight ratio: the ratio of LPS group was significantly higher(P<0.01).2)HE staining of lung tissue: the normal control group showed normal lung structure and clear alveolar cavity.The LPS group showed obvious lung damage,including destroyed alveolar structure,alveolar expansion,exudation,massive blood cell infiltration and capillary dilation.Both of TMP1 and TMP2 group had some degree of lung injury less than LPS group.3)The expression of protein on lung tissue: compared with the control group,the expression of ROCK2 and LIMK1 protein were up-regulated(P<0.05)and cofilin protein were down-regulated(P<0.05)in LPS group;compared with the LPS group,the expression of ROCK2,LIMK1 and cofilin proteins were no significance(P>0.05)in TMP1 group,the expression of LIMK1 protein was down-regulated(P<0.05)and ROCK2 and cofilin proteins had no difference(P>0.05)in TMP2 group.4)The results of immunofluorescence showed: compared with control group,the ECs showed obviously shrinking,ecreased volume and increased cells gap in LPS group;compared with LPS group,the ECs were observed significantly increased cell volume and decreased intercellular gaps,and the overall morphology and distribution of the four groups was significantly improved in the TMP1,TMP2 and TMP3 groups.The expression of protein on each group of ECs: compared with the control group,the expression of ROCK2,LIMK1 and p-cofilin protein were up-regulated(P<0.05),cofilin protein was down-regulated(P<0.05),and p-LIMK1 protein was no change(P>0.05)in LPS group;compared with LPS group,the expression of ROCK2,LIMK1 and p-cofilin protein were down-regulated(P<0.05),and p-LIMK1 protein was no difference(P>0.05)in TMP1,TMP2 and TMP3 group.Compared with control group,the relative expression of p-cofilin/cofilin was increased(P<0.05)in LPS group.Conclusion: 1)Tetramethylpyrazine can reduce LPS-induced acute lung injury;2)The mechanism of tetramethylpyrazine in reducing LPS-induced ALI may be related to the expression of ROCK2,LIMK1 and p-cofilin protein down-regulation,stabilization of cytoskeleton,and reduction of alveolar capillary permeability. |
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