| Objective The phenotype transformation of airway smooth muscle cells(ASMC)in asthma is an important mechanism involved in airway remodeling.It was found in our previous study that HMGB1 was involved in airway remodeling in bronchial asthma and the thickness of ASM around the airway was reduced in a mouse model of asthma after HMGB1 activity was antagonized.Inhibition of high mobility group box1 protein(HMGB1)in vivo and in vitro reduced the thickness of airway smooth muscle(ASM)around the trachea in mice,and the underlying mechanism,however,remains unclear.The present study was designed to preliminarily investigate the effect and related mechanisms of HMGB1 on the phenotype transformation of ASMCs in rats.Methods ASMCs derived from SD rats were cultured using primary cell culture method.Briefly,ASMCs were cultured in DMEM supplemented with 10% fetal bovine serum(FBS)for 24 hs and replaced with serum-free DMEM for extra 24 hrs to synchronize the cells.(1)ASMCs were divided into 4 groups,including 1 the control group,which was treated with DMEM supplemented with 2% serum alone;2 the HMGB1 100ng/ml group,which was treated with DMEM supplemented with 2% serum and 100ng/ml HMGB1;3 the HMGB1 500ng/ml group,which was treated with DMEM supplemented with 2% serum and 500ng/ml HMGB1;and 4 the HMGB1 1000ng/ml group,which was treated with DMEM supplemented with 2% serum and 1000ng/ml HMGB1.Cells in each group were seeded in 6-well plate and cultured for 24 hrs.After the treatment,these cells were digested with 0.25% trypsin and harvested for index analysis.(2)Effect of HMGB1 on the migration ability of ASMCs was determined by wound healing and Transwell assays.Effect of different concentrations of HMGB1 on the cytoskeleton rearrangement of ASMCs was observed by immunofluorescence,that on the cell cycle distribution was detected by flow cytometry,and that on the expression of proliferating cell nuclear antigen(PCNA),?-smooth muscle actin(?-actin),and SM-22? in ASMCs was determined by Western blot(WB).(3)Antibodies against receptor for advanced glycation end products(RAGE,20?g/ml),Toll like receptor-2(TLR2,20?g/ml)and Toll like receptor-4(TLR4,20?g/ml)were applied,respectively,in the HMGB1 1000ng/ml group,and the expression of ?-actin and SM-22? was determined by WB;the effect on cytoskeleton rearrangement of ASMCs was observed by immunofluorescence.Antibodies against RAGE(20?g/ml)and LY294002(10?M)were applied,respectively,and the expression level of phosphorylated AKT in ASMCs was detected by WB.Results Wound healing and Transwell assays showed that HMGB1 enhanced the migration ability of ASMCs and the migration of ASMCs increased with the increased concentration of HMGB1.Fluorescent microscopy observation indicated that HMGB1 stimulated the cytoskeleton rearrangement of ASMCs with increased amount of myofilaments.Most cells were plate-like with strong tension.The effect was enhanced as the increased concentration of HMGB1.When treated by LY294002 and anti-RAGE antibody,the effect of HMBG1 was decreased with reduced myofilaments.Effect of different concentrations of HMGB1 on cell cycle distribution of ASMCs was detected by flow cytometry and the results showed that treated with different concentrations of HMGB1,proportion of ASMCs at the G2 and S phases was significantly increased in the HMGB1 500ng/ml and HMGB1 1000ng/ml groups,which was statistically increased compared with that of the control group and the HMGB 100ng/ml group(p<0.05).No statistically significant difference in the proportion of cells at G2 and S phase was observed between the control group and the HMGB1 100ng/ml group(p>0.05).Expression of PCNA protein in ASMCs was significantly increased by HMGB1(p<0.05)and the expression increased significantly with increased concentration of HMGB1.WB results showed that the expression levels of ?-actin and SM-22? in ASMCs were significantly reduced by HMGB1(p<0.05),which were further significantly decreased with the increased concentration of HMGB1.After cells in the HMGB1 1000ng/ml group were treated with anti-RAGE(20?g/ml),anti-TLR2(20?g/ml),and antiTLR4(20?g/ml)antibodies,respectively,the expression levels of ?-actin and SM-22? in ASMCs of the anti-RAGE + HMGB1 1000ng/ml group were upregulated and statistically different compared with those of the HMGB1 1000ng/ml group,the anti-TLR2 and anti-TLR4 antibodies treated groups(p<0.05).No significant difference was noted among the latter three groups(p>0.05).After the phosphatidy linositol 3-kinase(PI3K)signal pathway was suppressed by LY294002,the expression levels of ?-actin and SM-22? in ASMCs were up-regulated as evidenced by WB,and the LY294002+ HMGB1 1000ng/ml group was statistically different versus the control group,the HMGB1 1000ng/ml group and the LY294002 alone group(p<0.05).No difference was observed among the latter three groups(p>0.05).Expression levels of phosphorylated AKT in the control group,the HMGB1 1000ng/ml group,the LY294002+HMGB1 1000ng/ml group and the anti-RAGE +HMGB1 1000ng/ml group were measured by WB and the results showed that the levels of phosphorylated AKT in the LY294002+HMGB1 1000ng/ml group and the anti-RAGE + HMGB1 1000ng/ml group were decreased compared with that of the control group and the HMGB11000ng/ml group(p<0.05).Conclusions HMGB1 induces the migration of ASMCs by altering the morphology and cytoskeleton recombination of the cells,enhancing the cell cycle and the expression of PCNA in cell cycle progression,and eventually promoting the cell proliferation.HMGB1 regulates the cell phenotypic and functional transformation via the RAGE and PI3 K signaling pathways instead of enhancing the expression of TLR2 or TLR4.HMGB1 promotes the phenotype transformation of ASMCs by upregulating the expression of RAGE receptor,which may provide a new theory for ASM remodeling in asthma.These results offer new insights into the mechanism of ASM remodeling and provide an effective treatment for airway remodeling in asthma.Part ?: P rimary Culture and Identification of SD Rat Airway Smooth Muscle CellsObjective To establish the primary culture method of SD rat airway smooth muscle cells(ASMCs)and to identify the cells,providing an experimental material for related research.Methods: 6-8-week old male healthy SD rats,weighing 200-250 g,were anaesthetized by intraperitoneal injection of 10% chloral hydrate and disinfected.Incision was made on the skin and subcutaneous tissue was separated to isolate the trachea.ASMCs were harvested by using the enzyme digestion method.Results: The cultured cells were identified by morphological observation and the expression of ?-actin determined through indirect immunofluorescence assay.Conclusion: high purity tracheal smooth muscle cells are obtained by enzyme digestion method.Part ?: Regulation of HMGB1 on the phenotype transformation of ASMCs.Objective To investigate the effect of different concentrations of HMGB1(0,100,500 ng,and 1000ng/ml)on the phenotype transformation of rat ASMCs.Methods:ASMCs were divided into 4 groups,including 1 the control group,which was treated with DMEM supplemented with 2% serum alone;2 the HMGB1 100ng/ml group,which was treated with DMEM supplemented with 2% serum and 100ng/ml HMGB1;3 the HMGB1 500ng/ml group,which was treated with DMEM supplemented with 2% serum and 500ng/ml HMGB1;and 4 the HMGB1 1000ng/ml group,which was treated with DMEM supplemented with 2% serum and 1000ng/ml HMGB1.Cells in each group were seeded in 6-well plate and cultured for 24 hrs.After the treatment,these cells were digested with 0.25% trypsin and harvested for index analysis.Effect of HMGB1 on the migration ability of ASMCs was determined by wound healing and Transwell assays.ASMCs were stained with TRITC-labelled phalloidin and observed under a fluorescence microscope to detect the effect of different concentrations of HMGB1 on the cytoskeleton rearrangement and myofilaments.Effec t of different concentrations of HMGB1 on the cell cycle distribution was determined by flow cytometry after PI staining,and that on the expression of PCNA,?-actin,and SM-22? in ASMCs were detected by WB.Results: Wound healing and Transwell assays showed that HMGB1 enhanced the migration ability of ASMCs and the migration of ASMCs increased with the increased concentration of HMGB1.In the wound healing assay,the repair area of ASMCs in each group was observed and calculated after 24 hrs of interventio n with different concentrations of HMGB1.The cell repair areas in the control group,the HMGB1 100ng/ml group,the HMGB1 500ng/ml group and the HMGB1 1000ng/ml group were 34.54±2.62%?48.32±3.15%?74.23±3.43%?83.65±4.21%,respectively.The repair areas in the HMGB1 100ng/ml group,the HMGB1 500ng/ml group and the HMGB1 1000ng/ml group were higher than that of the control group(p<0.05),and that of the HMGB1 500ng/ml group and the HMGB1 1000ng/ml group was increased compared with the control group and the HMGB1 100ng/ml group in a dose-dependent manner(p< 0.01).In Transwell assay,the number of cells migrated in the control group,the HMGB1 100ng/ml group,the HMGB1 500ng/ml group and the HMGB1 1000ng/ml group were 8.92±0.13?12.3±0.57?18.3±0.28 and 24.5±0.23,respectively.The number of migrated cells in the HMGB1 100ng/ml group,the HMGB1 500ng/ml group,and the 1000ng/ml HMGB1 group was increased compared with the control group(p<0.05),and that of the MGB1 500ng/ml group and the 1000ng/ml group was increased significantly compared with the control group and HMGB1 100ng/ml group in a dose-dependent manner(p<0.01).HMGB1 promoted the migration of ASMCs and the migration ability also increased with the increase of HMGB1 concentration.TRITC-labeled phalloidin staining was used to detect the configuration of ASMCs and the changes of the myofilaments and fluorescence microscopy showed HMGB1 stimulated the cytoskeleton rearrangement of ASMCs increased the content of myofilaments.Most cells were plate-like with strong tension.The effect was enhanced by the increased concentration of HMGB1.Effect of different concentrations of HMGB1 on cell cycle distribution was detected by flow cytometry after PI staining and the results showed that the proportion of cells at the G0/G1 phase in the control group,the HMGB1 100ng/ml,500ng/ml and 1000ng/ml groups were 83.83±2.62?79.31±1.35?73.24±2.23?65.37±1.81,respectively;those at the S phase were 5.62±0.31?7.19±0.52?14.93±1.27?15.92±1.33,respectively;those at the G2 phase were 8.14±0.45?9.37±0.63?13.36±0.95?19.91±1.54.The proportions of cells out the G1 phase and into the G2 and S phases in the HMGB1 500ng/ml group and HMGB1 1000ng/ml group were significantly increased compared with the control group and the HMGB1 100ng/ml group,with statistically significant difference(p<0.05).There was no significant difference between the control group and the HMGB1 100ng/ml group in terms of the proportion of cells at G2 and S phases(p>0.05).The results of Western blot showed that compared with the control group,the expression of SMA-22?and ?-SMA in the ASMCs of the HMGB1 100ng/ml,500ng/ml,and 1000ng/ml groups was significantly decreased(p<0.05),and the expression was negatively correlated with the dose of HMGB1.Compared with the control group,the level of PCNA was increased in the HMGB1 100ng/ml,500ng/ml and 1000ng/ml groups(p<0.05),which was positively correlated with the dose of HMGB1(p< 0.05).Conclusion: HMGB1 promotes the proliferation,migration,cytoskeleton rearrangement of ASMCs and increases the amount of myofilaments to participate in the phenotype transformation of ASMCs,and these effects of HMGB1 are enhanced as the concentration of HMGB1 increases.Part ?.Mechanisms of HMGB1 in regulating the phenotype transformation of ASMCs.Objective To investigats and observe the effect of receptor antagonists against RAGE,TLR2 and TLR4,and LY294002 on the HMGB1-induced phenotype transformation,cytoskeleton proteins,and phosphorylated AKT in ASMCs,and to explore the potential pathways involved in the regulatory effect of HMGB1.Methods: ASMCs in the HMGB1 1000ng/ml group were treated with antibodies against RAGE(20?g/ml),TLR2(20?g/ml),and TLR4(20?g/ml),respectively,the expression of ?-actin and SM-22? was determined by WB and the effect of HMGB1 on the cytoskeleton rearrangement was observed by fluorescence microscope;ASMCs in the HMGB1 1000ng/ml group were treated with LY294002(10?M),the expression of ?-actin and SM-22? was determined by WB,and the effect of HMGB1 on the cytoskeleton rearrangement was observed by fluorescence microscope;ASMCs in the HMGB1 1000ng/ml group were treated with RAGE(20?g/ml)and LY294002(10?M),respectively,and the expression of phosphorylated AKT was detected by WB.Results: After cells in the HMGB1 1000ng/ml group were treated with antiRAGE(20?g/ml),anti-TLR2(20?g/ml),and anti-TLR4(20?g/ml)antibodies,respectively,the expression levels of ?-actin and SM-22? in ASMCs of the antiRAGE+ HMGB1 1000ng/ml group were upregulated and statistically different compared with those of the HMGB1 1000ng/ml group,the anti-TLR2 and antiTLR4 antibodies-treated groups(p<0.05).No significant difference was noted among the latter three groups(p>0.05).After the phosphatidy linositol 3-kinase(PI3K)signal pathway was suppressed by LY294002,the expression levels of ?-actin and SM-22? in ASMCs were up-regulated,and the LY294002+ HMGB1 1000ng/ml group was statistically different versus the control group,the HMGB1 1000ng/ml group and the LY294002 alone group(p<0.05).No difference was observed among the latter three groups(p>0.05).Expression level of phosphorylated AKT in the control group,the HMGB1 1000ng/ml group,the LY294002+HMGB1 1000ng/ml group and the anti-RAGE +HMGB1 1000ng/ml group was detected by WB and the results showed that the levels of phosphorylated AKT in the LY294002+HMGB1 1000ng/ml group and the antiRAGE + HMGB1 1000ng/ml group were decreased compared with that of the control group and the HMGB11000ng/ml group(p<0.05).Application of LY294002 and anti-RAGE antibody attenuated the effect of HMBG1 on the cytoskeleton rearrangement of ASMCs and reduced the amount of myofilaments.HMBG1 could induce the phosphorylation and activation of AKT and significantly increase the expression of P-AKT,which was inhibited by LY294002.Conclusion: HMGB1 regulates the cell phenotypic and functional transformation via the RAGE and PI3 K signaling pathways instead of enhancing the expression of TLR2 or TLR4.HMGB1 promotes the phenotype transformation of ASMCs by upregulating the expression of RAGE receptor,which may provide a new theory for ASM remodeling in asthma.These results offer new insights into the mechanism of ASM remodeling and provide an effective treatment for airway remodeling in asthma. |
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