| Objective:Acute lung injury（ALI）is a common disease with high morbidity and mortality.Although some progress has been made in clinical treatment,the mortality is still not low.Therefore,it is of great clinical significance to further reveal the pathogenesis of ALI.Currently,A large number of studies have revealed that lipolyaccharide（LPS）is the main component of the outer membrane of Gram-negative bacteria,which plays a key role in inducing inflammation and leading to lung injury.Therefore,LPS has been used to induce ALI animal and cell models.In addition,as impaired proliferation of alveolar type II epithelial cells is a key pathological change of ALI.So the purpose of this study was to examine the role of SMYD2 in LPS-induced proliferation of ALI alveolar type II epithelial cells and its potential molecular mechanism at the animal and cellular level.In order to lay an experimental foundation for revealing the pathogenesis of ALI comprehensively and provide a new perspective for the clinical development of new effective drugs to treat ALI.Methods:一、Firstly,LPS was used to treat MLE-12 cells to construct ALI cell model in vitro,namely control group and LPS group.CCK-8 was used to detect the viability of MLE-12cells,and Real-time PCR was used to detect SMYD2,TCF-1 and SPC m RNA expressions in MLE-12 cells.The protein expressions of SMYD2,TCF-1 and SPC in MLE-12 cells were detected by Western blot.The correlation between SMYD2expression and ALI type II epithelial cell proliferation was evaluated in vitro.Then,LPS was used to intervene C57BL/6 mice to construct ALI models in vivo,namely the control group and LPS group.HE was used to observe the pathological changes,TUNEL staining was used to observe the apoptosis of lung tissue cells,ELISA was used to detect the contents of total protein in alveolar lavage solution,blood gas analyzer was used to observe the concentration of blood oxygen,and the dry-wet ratio of lung tissue was measured.Primary alveolar type II epithelial cells were then isolated,and CCK-8 was used to detect the viability of primary alveolar type II epithelial cells.m RNA expressions of SMYD2,TCF-1 and SPC in primary alveolar type II epithelial cells were detected by Real-time PCR.The expressions of SMYD2,TCF-1 and SPC in primary alveolar type II epithelial cells were detected by Western blot.The correlation between SMYD2expression and ALI type II epithelial cell proliferation was evaluated in vivo.For subsequent research SMYD2 role in ALI and mechanism experimental basis.二、SMYD2 overexpressing adenovirus（Ad-SMYD2）was used to up-regulate the expression of SMYD2 in MLE-12 cells,Ad-β-Gal was used as a negative control,and SMYD2 silencing adenovirus（Ad-sh-SMYD2）was used to down-regulate the expression of SMYD2 in MLE-12 cells.Ad-sh-NC was used as negative control.After verifying the infection efficiency,ALI cell model was constructed in vitro.The activity of MLE-12 cells was detected by CCK-8,and the m RNA expressions of SMYD2,SPC,CCND1 and c-Myc in MLE-12 cells were detected by Real-time PCR.Western blot was used to detect the expression of SMYD2,TCF-1,β-catenin and me-β-catenin in the cytoplasm and nucleus of MLE-12 cells,and immunofluorescence was used to observe the expression and localization of SMYD2,TCF-1,β-catenin and SPC in MLE-12 cells.In addition,after inhibiting the enzyme activity of SMDY2 with LLY-507,the expressions of total SMYD2,TCF-1,β-catenin and me-β-catenin were detected by Western blot.Meanwhile,the effect of SMYD2 in ALI on TCF-1’s regulation of CCND1and c-Myc promoter activity was proved by double luciferase assay,and the effect of SMYD2 in ALI on TCF-1 binding CCND1 and c-Myc promoter was observed by Ch IP assay.The Co-IP assay was used to analyze the effect of SMYD2 on the interaction between FOXM1 andβ-catenin in ALI.At the cellular level,it was demonstrated that SMYD2 reversed the damage effect of LPS on MLE-12 cell proliferation by promotingβ-catenin methylation and activatingβ-catenin/TCF-1 pathway.三、The in vivo model of ALI was established by LPS intervention in C57BL/6mice.And Use Ad-SMYD2,Ad-β-Gal,Ad-sh-SMYD2 or Ad-sh-NC regulate the expression of SMYD2 in mouse lung tissue.Then HE was used to observe the pathological changes,TUNEL staining was used to observe the apoptosis of lung tissue cells,ELISA was used to detect the content of total protein and albumin in alveolar lavage fluid,blood gas analyzer was used to observe the concentration of blood oxygen,and the dry-wet ratio of lung tissue was measured.The expression and distribution of SMYD2,TCF-1,β-catenin and SPC in lung tissues were detected by immunofluorescence staining.To evaluate the role and potential mechanism of SMYD2in the characterization and pathological changes of LPS induced ALI disease.Primary alveolar type II epithelial cells were then isolated and cultured.CCK-8 was used to detect the viability of primary alveolar type II epithelial cells,and Real-time PCR was used to detect the m RNA expression of SMYD2,SPC,CCND1 and c-Myc in primary alveolar type II epithelial cells.The expressions of SMYD2,TCF-1,β-catenin and me-β-catenin in the cytoplasm and nucleus of primary alveolar type II epithelial cells were detected by Western blot.In addition,the expression of total SMYD2,TCF-1,β-catenin and me-β-catenin in lung tissues was detected by Western blot after LLY-507 was used to inhibit the enzyme activity of SMDY2 in vivo.It was demonstrated in vitro that SMYD2reversed the damage of LPS on the proliferation of mouse alveolar type II epithelial cells byβ-catenin methylation and activatingβ-catenin/TCF-1pathway.Result:一、Compared with the control group,the motility of MLE-12 cells in LPS group was significantly decreased,the m RNA expression levels of SMYD2,TCF-1 and SPC were significantly decreased,and the protein expression levels of SMYD2,TCF-1 and SPC were also significantly decreased（P<0.05）.Compared with the control group,the lung tissue structure of mice in LPS group was damaged and there was a large number of inflammatory cell infiltration,the apoptosis of lung tissue was significantly increased,the contents of total protein in alveolar lavage fluid of mice were significantly increased,the concentration of Pa O2 in blood was significantly decreased,and the dry-wet ratio of lung tissue was significantly increased（P<0.05）.Compared with the control group,the vitality of primary alveolar type II epithelial cells in LPS group was significantly decreased,m RNA expression levels of SMYD2,TCF-1 and SPC were significantly down-regulated,and protein expression levels of SMYD2,TCF-1 and SPC were also significantly down-regulated（P<0.05）.二、Overexpression of SMYD2 could promote the viability of MLE-12 cells and reverse the inhibitory effect of LPS on the viability of MLE-12 cells.Overexpression of SMYD2 can promote the expression of SPC,CCND1 and c-Myc m RNA,and reverse the inhibitory effect of LPS on the expression of these indicators.Overexpression of SMYD2can promote the expression of TCF-1 in the nucleus,and reverse the inhibitory effect of LPS on the expression of TCF-1 in the nucleus.Overexpression of SMYD2 can promote the methylation ofβ-catenin and transfer it to the nucleus,and reverse the inhibition of LPS on the methylation ofβ-catenin and block the cytoplasm.Vice versa,silencing SMYD2 inhibited the viability of MLE-12 cells and promoted the inhibitory effect of LPS on the viability of MLE-12 cells.Silencing SMYD2 could inhibit the expression of SPC,CCND1 and c-Myc m RNA,and enhance the down-regulation effect of LPS on the expression of these indicators.Silencing SMYD2 inhibited the expression of TCF-1 in the nucleus,and further enhanced the inhibitory effect of LPS on the expression of TCF-1 in the nucleus.Silencing SMYD2 inhibitedβ-catenin methylation and enhanced LPS inhibitedβ-catenin methylation to block cytoplasmic function.In addition,overexpression of SMYD2 activated theβ-catenin/TCF-1 signaling pathway and reversed the inhibition of LLY-507 on theβ-catenin/TCF-1 pathway.Meanwhile,the effect of SMYD2 in ALI on TCF-1’s regulation of CCND1 and c-Myc promoter activity was proved by double luciferase assay,and the effect of SMYD2 in ALI on TCF-1binding CCND1 and c-Myc promoter was observed by Ch IP assay.The Co-IP assay was used to analyze the effect of SMYD2 on the interaction between FOXM1 andβ-catenin in ALI.At the cellular level,it was demonstrated that SMYD2 reversed the damage effect of LPS on MLE-12 cell proliferation by promotingβ-catenin methylation and activatingβ-catenin/TCF-1 pathway.三、Compared with the LPS-Ad-β-Gal group,the damage of lung tissue structure and inflammatory cell infiltration were alleviated in the LPS-Ad-SMYD2 group,the apoptosis of lung tissue cells was significantly reduced,the contents of total protein and albumin in the alveolar lavage fluid of mice were significantly decreased,the concentration of Pa O2 in the blood of mice was significantly increased and the concentration of Pa CO2 was significantly decreased.The dry-wet ratio of lung tissue was significantly decreased（P<0.05）,and the fluorescence signals of SMYD2,TCF-1 and SPC were enhanced,while the fluorescence signals ofβ-catenin were not significantly changed.Compared with the LPS-Ad-β-Gal group,the activity of primary alveolar type II epithelial cells in the LPS-Ad-SMYD2 group was significantly increased,and m RNA expression levels of SMYD2,TCF-1 and SPC were significantly up-regulated.The protein expressions of SMYD2 and me-β-catenin in cytoplasm were up-regulated while the protein expressions ofβ-catenin were down-regulated.The protein expressions of SMYD2,TCF-1,me-β-catenin and me-β-catenin in nucleus were significantly up-regulated（P<0.05）.Compared with the LPS-Ad-sh-NC group,the lung tissue structure of mice in the LPS-Ad-sh-SMYD2 group was more severely damaged and a large number of inflammatory cells infiltrated,the lung tissue cell apoptosis was significantly increased,the contents of total protein and albumin in the alveolar lavage fluid of mice were significantly increased,the concentration of Pa O2 in the blood of mice was significantly decreased and the concentration of Pa CO2 was significantly increased.The dry-wet ratio of lung tissue in mice was significantly increased（P<0.05）.The fluorescence signals of SMYD2,TCF-1 and SPC were weakened,while the fluorescence signals ofβ-catenin were not significantly changed.Compared with the LPS-Ad-sh-NC group,the activity of primary alveolar type II epithelial cells in the LPS-Ad-sh-SMYD2group was significantly decreased,and the m RNA expression levels of SMYD2,TCF-1and SPC were significantly down-regulated.The expression of SMYD2 and me-β-catenin protein was down-regulated and the expression ofβ-catenin protein was up-regulated in the cytoplasm.The expression of SMYD2,TCF-1,me-β-catenin andβ-catenin in the nucleus were significantly down-regulated（P<0.05）.In addition,compared with the LPS-Ad-SMYD2-DMSO group,the expression of SMYD2 in the LPS-Ad-SMYD2-LLY507 group was no significantly change,and the expression of TCF-1 and me-β-catenin proteins were down-regulated（P<0.05）,otherwise the expression ofβ-catenin protein was no significantly change（P>0.05）.Conclusion:1.LPS can induce the pathological changes of ALI in vivo and in vitro,and can be used as an effective method for ALI models.2.In LPS-induced ALI in vivo and in vitro,the expression level of SMYD2 and TCF-1 were significantly down-regulated.3.SMYD2 can improve LPS-induced inhibition of MLE-12 cell proliferation.4.SMYD2 can methylateβ-CATENIN to activateβ-Catenin/TCF-1 signaling pathway plays a role in improving LPS-induced proliferation inhibition of MLE-12 cells.5.SMYD2 can improve LPS-induced acute lung injury in mice.6.SMYD2 can activateβ-Catenin/TCF-1 signaling pathway to attenuate alveolar type II epithelial cell injury in ALI mice. |
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