Research On The Mechanism Of Shegan Mahuang Decoction Inhibiting The Proliferation Of Smooth Muscle Based On TRPV1/TAS2R14 Activating Bcl-2/Bnip3 Mediated Mitochondrial Activity

Objective:The bronchial asthma rat model was established by Ovalbumin（OVA）sensitization and the proliferation model of airway smooth muscle cells（ASMCs）induced by platelet derived growth factor-BB（PDGF-BB）in vitro.Based on the inhibition of smooth muscle proliferation mediated by B-cell lymphoma-2（Bcl-2）/Bcl-2 nineteen kilodalton interacting protein 3（Bnip3）activated by Transient Receptor Potential Vanilloid-1（TRPV1）/Taste Receptor Type 2 Member 14（TAS2R14）,the molecular mechanism of Shegan Mahuang Decoction（SMD）on bronchial asthma airway remodeling was studied.It is expected to take SMD as an example to explain the scientific connotation of"Xin Xuan Ku Xie".MethodsTheoretical research part:Through sorting out the discussion on the naming,etiology,pathogenesis and treatment prescriptions of asthma in ancient Chinese medicine books,this paper summarizes the common treatment methods of the basic pathogenesis of asthma.And collect the modern research literature on the pungent and bitter taste of traditional Chinese medicine,and explain the correlation between the treatment of asthma"Xin Xuan Ku Xie"and TRPV1 and TAS2R14.Experimental research part:Experiment 1 Effect of Shegan Mahuang Decoction on Ova induced asthmatic rats Seventy SPF grade male SD rats were randomly divided into 7 groups with 10 rats in each group.In addition to the normal group,ova1ml was injected to sensitize the model.The control group and model group were given distilled water solution of equal volume by gavage,dexamethasone（DEX）group 0.405mg/kg/d,Guilong Kechuanning（GK）group 0.405g/kg/d,Shegan Mahuang Decoction high,medium and low dose groups12.96,6.48 and 3.24g/kg/d by gavage.The lung injury of rats in each group was observed by hematoxylin eosin staining.Serum TSLP,IL-5,IL-13 and TNF-αwere detected by ELISA,The m RNA expression levels of TRPV1,TAS2R14,Bcl-2,Bnip3,caspase-3 and Cyt C were detected by RT-q PCR,and the protein expression levels of TRPV1,TAS2R14,Bcl-2,Bnip3,caspase-3 and Cyt C were detected by WB.Experiment 2 Effect of SMD medicated serum on the proliferation of ASMCs stimulated by PDGF-BB by inducing Bcl-2/Bnip3 activation through TRPV1/TAS2R14The serum containing Shegan Mahuang Decoction was prepared,and the best concentration and action time were selected by CCK8.The proliferation model of ASMCs stimulated by PDGF-BB was constructed.Control group:routine cell culture.PDGF-BB group:PDGF-BB 10 ng/ml stimulation.SMD medicated serum group:on the basis of PDGF-BB group,it was treated with medicated serum containing 10%SMD.Agonist group:capsaicin 500 nmol/L（chloroquine,1 mmol/L）intervention.Inhibitor group:AMG9810 10umol/L（5’-AMP,5mmol/L）intervention.SMD medicated serum+agonist group:10%SMD medicated serum+capsaicin 500nmol/L（chloroquine,1mmol/L）intervention.SMD drug containing serum+inhibitor group:10%SMD drug containing serum+AMG9810 10umol/L（5’-AMP,5mmol/L）intervention.It was cultured in a cell incubator with 5%CO₂at 37℃for 48h.The proliferation activity and cell cycle were detected by flow cytometry.Experiment 3 Effect of SMD medicated serum on mitochondrial activity of ASMCs stimulated by PDGF-BB by regulating Bcl-2/Bnip3ASMCs were divided into control group:ASMCs were stimulated with PDGF-BB 10ng/ml.si-Bcl-2 group:ASMCs transfected with si-Bcl-2 were stimulated by PDGF BB10 ng/ml.SMD group:PDGF-BB was stimulated with 10 ng/ml and intervened with10%SMD containing serum.SMD+si-Bcl-2（Bnip3）group:PDGF-BB 10 ng/ml stimulated Bcl-2（Bnip3）si RNA transfection into ASMC,and then intervened with 10%SMD containing serum.The changes of mitochondrial ATPase activity and mitochondrial membrane potential were detected.ResultsTheoretical research:The imbalance of lung qi is the basic pathogenesis of asthma,and Xin Xuan Kuxie is its fundamental treatment.The exertion of pungent and bitter drugs is related to TRPV1and TAS2R14 respectively.SMD is not only a classic formula for the treatment of asthma,but also a representative formula of the compatibility of Xinxuan and Kuxie.Therefore,it is possible to treat asthma by adjusting TRPV1 and TAS2R14.Experimental research:Experiment 1 Effect of Shegan Mahuang Decoction on Ova induced asthmatic rats The infiltration of bronchitis cells and the destruction of lung tissue structure were alleviated in SMD-L group and SMD-H group.In SMD-M group,DEX group and GK group,the infiltration of inflammatory cells in blood vessels and peribronchial areas decreased,the thickening of airway smooth muscle was not obvious,and the bronchial structure was still intact.Compared with the model group,IL-5,IL-13 and TNF-αin each treatment group There was no significant difference in the reduction of TSLP in SMD-L group,but there were differences in other treatment groups（P<0.01）.The expression levels of TRPV1 m RNA（P<0.01）,Bnip3 m RNA（P<0.05）,caspase-3m RNA（P<0.01）and Cyt C m RNA（P<0.01）decreased in SMD-L group,but there was no significant difference in the expression levels of TAS2R14 m RNA and Bcl-2m RNA.In SMD-M group,the expression levels of TRPV1 m RNA,Bnip3 m RNA,caspase-3 m RNA and Cyt C m RNA were significantly decreased（P<0.01）,while the expression levels of TAS2R14 m RNA and Bcl-2 m RNA were significantly increased（P<0.01）.In SMD-H group,TRPV1 m RNA,Bnip3 m RNA,caspase-3 m RNA and Cyt C m RNA were significantly down regulated（P<0.01）,while the expression levels of TAS2R14 m RNA and Bcl-2 m RNA were significantly up regulated（P<0.01）.In SMD-L group,the expression levels of TRPV1（P<0.05）,Bnip3（P<0.05）and Cyt C（P<0.01）were down-regulated,and the expression levels of Bcl-2（P<0.01）were up-regulated.There was no significant difference between TAS2R14 and caspase-3.In SMD-M group,the expression levels of TRPV1,Bnip3,caspase-3 and Cyt C protein were significantly down regulated（P<0.01）,while the expression levels of TAS2R14and Bcl-2 were significantly up regulated（P<0.01）.In SMD-H group,TRPV1,Bnip3,caspase-3 and Cyt C proteins were significantly down regulated（P<0.01）,while the expression levels of TAS2R14 and Bcl-2 proteins were significantly up regulated（P<0.01）.Experiment 2 Effect of Shegan Mahuang Decoction medicated serum on theproliferation of ASMCs stimulated by PDGF-BB by inducing Bcl-2/Bnip3activation through TRPV1/TAS2R14The optimum action time and concentration of Shegan Mahuang Decoction containing serum were 10%and 48 hours respectively.Compared with PDGF-BB group,the cell proliferation activity of PDGF-BB+SMD group,PDGF-BB+AMG9810 group,PDGF-BB+capsaicin+SMD group and PDGF-BB+AMG9810+SMD group decreased（P<0.01）,while that of PDGF-BB+capsaicin group increased（P<0.01）.The cell proliferation activity decreased in PDGF-BB+chloroquine group and PDGF-BB+chloroquine+SMD group（P<0.01）,and increased in PDGF-BB+5’-amp group and PDGF-BB+5’-amp+SMD group（P<0.01）.In PDGF-BB+SMD group（P<0.05）,PDGF-BB+AMG9810 group（P<0.01）and PDGF-BB+AMG9810+SMD group（P<0.01）,the number of G1 phase cells decreased and the number of G2phase cells increased（P<0.01）.In PDGF-BB+capsaicin group,the number of G1phase cells increased（P<0.01）,and the number of G2 phase cells decreased（P<0.01）.Compared with PDGF-BB+capsaicin+SMD group,there was no significant difference in the number of G1 phase cells,and the number of G2 phase cells decreased（P<0.01）.In PDGF-BB+SMD group,PDGF-BB+chloroquine group and PDGF-BB+chloroquine+SMD group,the number of G1 phase cells decreased（P<0.05）,and the number of G2 phase cells increased（P<0.01）.In PDGF-BB+5′-AMP group,the number of G1 phase cells increased（P<0.05）,and the number of G2 phase cells decreased（P<0.01）.Compared with PDGF-BB+5′-AMP+SMD group,there was no significant difference in the number of G1 cells,and the number of G2 cells decreased（P<0.05）.There was no significant difference in the number of S-phase cells between the two groups.Experiment 3 Effect of Shegan Mahuang Decoction containing serum on mitochondrial activity of ASMCs stimulated by PDGF-BB by regulating Bcl-2/Bnip3Compared with the control group,the activity of Na⁺-K⁺-ATPase in si-Bcl-2 group increased significantly（P<0.01）,and there was no significant difference in Ca²⁺-Mg²⁺-ATPase.The activity of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase in SMD group and SMD+si-Bcl-2 group decreased（P<0.01,P<0.05）.The activities of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase decreased significantly in Si-Bnip3 group,SMD group and SMD+Si-Bnip3 group（P<0.01）.PE/FITC decreased significantly in all groups（P<0.01）.Conclusion1.Shegan Mahuang Decoction can reduce airway remodeling and reduce IL-5,IL-13and TNF-αin rats,Expression of TSLP and other remodeling related factors.The mechanism may be related to the expression of Bcl-2 and Bnip3 regulated by TRPV1and TAS2R142.Shegan Mahuang Decoction can inhibit the proliferation of rat ASMCs induced by PDGF-BB.Its mechanism may be related to increasing Bcl-2 and reducing the expression of Bnip3,inhibiting ATPase activity and reducing mitochondrial membrane potential,limiting mitochondrial activity,inhibiting ASMCs proliferation activity and blocking cell cycle.3.The mechanism of Shegan Mahuang Decoction of Xinxuan Kuxie treatment may be closely related to the activation of Bcl-2/Bnip3 mediated mitochondrial activity by TRPV1/TAS2R14.