| ObjectiveThe development of a rat model of chronic asthma was accomplished by stimulating ovalbumin（OVA）in vivo,and establishing a human bronchial epithelioid cell model（HBE）induced by transforming growth factor（TGF-β1）was also performed.From the perspective of autophagy,miRNA328-3p regulates the HMGB1 pathway,which may be implicated in the molecular mechanism of a prescription for Yanghe Pingchuan granules（YHPC）in bronchial asthma.Methods（1）The first experiment:A combination of OVA and aluminum hydroxide was used to develop the asthma rat models.Groups were divided into a normal group,a model group,Yanghe Pingchuan granules group(7.74g·kg-1),HMGB1 inhibitor group(0.01g·kg-1),Yanghe Pingchuan granules and HMGB1 inhibitor group(7.74g·kg-1+0.01g·kg-1).For four weeks,stimulation and intervention were followed by treatment.An analysis of pathological trachea morphology and airway mucus reserve of lung tissue in rats was performed using HE and PAS staining.Changes in the lung function and inflammatory cell count in bronchoalveolar lavage fluid（BALF）of rats.Cells from rat lung tissue were examined with transmission electronmicroscopy（TEM）to determine how the autophagic bodies form.The detection of IL-4,IL-13,CCR4,CXCR3 levels in rat serum by ELISA method.The detection of HMGB1,TLR4,IκB,and NFκBmRNA transcription levels using PCR.Using WB to detect HMGB1,LC3B,ATG5,α-SMA expression.By using IF,we were able to detect expression levels for TLR4,My D88,Beclin1,α-SMA.（2）The second experiment:By using CCK8 technique,an optimal concentration of TGF-β1 was found to stimulate bronchial epithelial cells,and the optimal concentration of Yanghe Pingchuan granules medicated serum and 3-MA was determined to intervene bronchial epithelial cells.After 48h of cell culture,the cells were divided into five groups:normal group,model group,Yanghe Pingchuan granules medicated serum group（YHPC group）,3MA group,Yanghe Pingchuan granules medicated serum and 3MA group（YHPC+3MA group）.Apoptosis was detected by FCM.TNF-α,IFN-γ,and IL-1βwas detected by ELISA.The markers of autophagy were observed under TEM.A PCR assay was used to detect the transcription and expression of HMGB1mRNA and miR328-3p.IF and WB were used to detect HMGB1,α-SMA,LC3B,ATG5,Beclin1,and p62 relative expression.（3）The third experiment:The purpose of this study is to design,synthesize,and screen a HMGB1 small interfering RNA that is silenced in the bronchial epithelial cells and transfected into them.Cell culture after 48 hours,each group was divided into six:a normal group,a model group,Yanghe Pingchuan granules medicated serum group（YHPC group）,TGF-β1 and si RNA-HMGB1-NC group（si RNA-HMGB1-NC group）,si RNA-HMGB1 group,Yanghe Pingchuan granules medicated serum and si RNA-HMGB1 group（YHPC+si RNA-HMGB1group）.Doubleluciferase was used to detect the miRNA328-3p and HMGB1 interactions.The expression levels of TNF-α,IFN-γ,and IL-1βwas detected by ELISA.FCM was used to detect apoptosis and TEM observed autophagy marker structure.Through the use of PCR,HMGB1mRNA were detected,relative expression of Beclin1,ATG5,p62,HMGB1,and LC3B identified by IF and WB methods.Results（1）First experiment:（1）According to HE and PAS staining,the lungs of rats in the model group were damaged compared with the normal group,producing excessive amounts of inflammatory cells and secretions,excessive metaplasia of airway goblet epithelial cells,thickening of smooth muscle layer,and increasing mucus secretion.In comparison with the model group,there was a reduction in inflammation from the trachea wall,a reduction in airway mucus hypersecretion,and a decrease in complications.As a result of Yanghe Pingchuan granules treatment,there was a reduction in inflammation and smooth muscle thickening around the trachea wall.（2）Based on Giemsa staining and lung function results,the model group had significantly more inflammatory cells in BALF than the normal group（P<0.01）.There was a decrease in the number of Eos,Lym,and Neu of inflammatory cells in the Yanghe Pingchuan granules combined HMGB1 inhibitor group and the HMGB1inhibitor group compared to the model group（P<0.05,P<0.01）.In the model group,the FVC,FEV0.3,and FEV0.3/FVC lung function indices decreased significantly compared with those in the normal group（P<0.01）.It was significant that the indexes of FVC,FEV0.3,and FEV0.3/FVC of lung function were higher in Yanghe Pingchuan granules group when compared with the model group（P<0.05,P<0.01）.（3）According to ELISA detections,rats in the model group,Yanghe Pingchuan group,and H inhibitor group had higher levels of IL-4,IL-13,CXCR3 and CCR4 in their serum compared to the normal group（P<0.05,P<0.01）,the IFN-γexpression was decreased（P<0.01）.A significant reduction in serum levels of IL-4,IL-13,CXCR3 and CCR4 was detected in rats with Yanghe Pingchuan treatment,H inhibitor therapy group,and Yanghe and HMGB1 inhibitory therapy compared with the model group（P<0.01）,the IFN-γexpression increased（P<0.01）.（4）Comparing the model group with the normal group,TEM analysis showed that the model group’s airway epithelial cells showed structural disorder.There was a decrease in autophagic bodies and autophagic vesicles in the Yanghe Pingchuan group and the H inhibitor group compared with the model group.（5）Compared with normal group,model group lung tissue showed significantly higher fluorescence expression of TLR4,My D88,Beclin-1,andα-SMA protein by IF,WB,and PCR（P<0.05,P<0.01）.Protein levels of TLR4,My D88,Beclin-1,andα-SMA remained significantly lower in the Yanghe Pingchuan group,the HMGB1 inhibitor group and Yanghe combined HMGB1 inhibitory therapy group than those in the model group（P<0.01）.HMGB1,TLR4,NFκBmRNA levels in the model group were higher than the normal groups（P<0.01）.As a result of this study,the levels of IκBmRNA decreased（P<0.01）.A decrease in HMGB1,TLR4,NFκBmRNA expression was observed in the lung tissue of the Yanghe Pingchuan group when compared with the model group（P<0.01）.The expression of IκBmRNA increased（P<0.01）.In the model group,the expression of LC3B,α-SMA,HMGB1,and ATG5 increased compared with the normal group（P<0.01）.Compared with the model group,the protein expression of LC3B,α-SMA,HMGB1,ATG5 decreased in the Yanghe Pingchuan group and Yanghe combined HMGB1 inhibitory therapy group（P<0.01,P<0.05）.（2）Second experiment:（1）According to the CCK8 test,compared to the blank group,cell viability increased after treatment with TGF-β1.The cell viability of 10μg/m L,20μg/m L,and 30μg/m L TGF-β1 was significantly higher than that of 5μg/m L and cell viability was significantly increased after adding 10μg/m L TGF-β1.Compared with the control group,all concentrations of Yanghe Pingchuan drug-containing serum at 24h had the most significant inhibitory effect on cells.With the increase of the concentration of drug-containing serum,the inhibitory effect become significant in a dose-dependent manner（P<0.05,P<0.01）.（2）Based on TEM and ELISA,compared with the normal group,structural disorders of airway epithelial cells were broken in the model group.Yanghe medicated serum group and 3-MA group showed a less autophagic body and autophagic vesicle number than the model group.It has been found that there is a significant difference between the model group and the normal cell group about the expression level of IL-1β,TNF-αcytokines（P<0.01）,IFN-γexpression prominently reduced.With Yanghe medicated serum treatment,the expression level of IL-1β,TNF-αcytokines was significantly reduced compared with the model group,IFN-γexpression significantly increased（P<0.01）.（3）The results of IF,WB and PCR showed that compared with the normal group,10μg/m L TGF-β1 the expression of HMGB1 and LC3B fluorescent protein increased in the model group（P<0.01）.Compared with the model group,the expression level of HMGB1 and LC3B fluorescent protein in Yanghe drug-containing serum treatment group decreased significantly（P<0.01）.As compared to a control group,10μg/m L TGF-β1 the relative expression of HMGB1,Beclin1,α-SMA and ATG5 protein in the treatment model group increased significantly（P<0.01）,and the relative expression of p62 protein decreased significantly（P<0.01）.In comparison with the normal group,the relative expression of HMGB1,Beclin1,α-SMA and ATG5 protein in Yanghe drug-containing serum treatment group decreased significantly,and the expression of p62 increased significantly（P<0.01）.To a compared normal group,10μg/m L TGF-β1the expression level of HMGB1mRNA in the model group was significantly increased,and the relative expression level of miR328-3p gene transcription was significantly decreased（P<0.01）.Compared with the model group,the transcription level of HMGB1mRNA gene in Yanghe drug-containing serum treatment group decreased significantly,and the transcription of miR328-3p increased significantly（P<0.01）.（4）The results of FCM showed that in contrast to the normal group,the model group showed significantly lower apoptosis rates（P<0.01）.In both the Yanghe medicated serum group and the 3-MA+Yanghe medicated serum group,the apoptosis rate was significantly higher than what was observed in the model group（P<0.01）.（3）Third experiment:（1）CCK8 detection and the double luciferase report showed that compared with the si RNA-NC,the transcription level of si RNA1,si RNA2,and si RNA3 HMGB1mRNA was inhibited（P<0.01,P<0.05）.Compared with the si RNA1,the transcription inhibition level of si RNA2 and si RNA3 HMGB1mRNA decreased（P<0.01,P<0.05）,and si RNA2 inhibition was the most significant.Compared with the normal group,hsa-miR-328-3P significantly down-regulated the expression of luciferase in HMGB1-3UTR-wt（P<0.01）.（2）According to TEM analyses,the structure of the epithelial cells in the model group was disrupted and autophagic bodies were increased when compared with the normal group.The number of autophagic bodies and autophagic vesicles in the YHPC group and si RNA-HMGB1 group was lower than that in model group.（3）The results of ELISA and PCR showed that compared with the normal group,IL-1β,TNF-αexpression levels were significantly increased,IFN-γwas decreased remarkably in the model group（P<0.01）.Compared with the model group,the IL-1β,TNF-αlevels were decreased significantly in the YHPC group,si RNA-HMGB1group,and YHPC+si RNA-HMGB1 group,the IFN-γexpression levels was notably increased（P<0.01）.In comparison with the normal group,the expression level of HMGB1mRNA in the model group was increased importantly（P<0.01）.Compared with the model group,the relative expression level of HMGB1mRNA gene transcription in the YHPC group,si RNA-HMGB1 group and YHPC+si RNA-HMGB1 group was decreased remarkably（P<0.05,P<0.01）.（4）The results of IF and WB showed that as compared to the normal group,the fluorescence expression of HMGB1 and LC3B increased significantly in the treatment group with 10μg/m L TGF-β1（P<0.01）.Compared with the model group,the expression level of HMGB1 and LC3B fluorescent protein in the treatment group with Yanghe medicated serum decreased significantly（P<0.01）.As compared to a control group,the expression of ATG5,Beclin1,and LC3B protein increased and the expression of p62 decreased in the treatment group with 10μg/m L TGF-β1（P<0.05,P<0.01）.Compared with the model group,the expression of ATG5,Beclin1 and LC3B protein in Yanghe drug-containing serum treatment group decreased significantly,and the expression of p62 increased（P<0.05,P<0.01）.（5）FCM detection showed that compared with the normal group,the apoptosis rate of the model group was importantly lower（P<0.01）.Compared with the model group,the apoptosis rate of the si RNA-HMGB1 group,the Yanghe medicated serum group,and the YHPC+si RNA-HMGB1 group increased importantly（P<0.01）.Conclusion1.There were various inflammatory cell infiltration in lung tissue and bronchi of asthmatic model rats,excessive tracheal mucus and collagen deposition led to HMGB1over-expression,which activated autophagy by mediating Beclin1,promoted the formation of autophagosomes,inhibited Th1 cell proliferation,promoted Th2 cell differentiation,and aggravated the progress of airway remodeling.2.The knockdown of HMGB1 expression significantly inhibits TGF-β1-induced fibrosis formation and inflammatory response in airway epithelial cells,promotes miR328-3p expression,inhibits downstream signaling pathway,blocks binding with Beclin-1,down-regulates autophagy activity,and promotes apoptosis.3.Yanghe Pingchuan granules can increase the expression level of miR328-3p,reduce the high level of extracellular HMGB1,inhibit the autophagy flow of airway epithelial cells,increase the rate of apoptosis,and thus reduce the increase of collagen synthesis and fibrosis.4.Yanghe Pingchuan granules can inhibit the HMGB1 expression,down-regulate the protein activity of the downstream signaling pathway,inhibit autophagy,increase Th1-related factor,enhance the differentiation of Th2 into Th1,reduce theα-SMA expression,and improve airway remodeling. |
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