The Phosphorylation Mechanism Of Acupuncture Relax Airway Smooth Muscle Based On The Metallothionein-2 In Asthma

Objective:Our previous study found that acupuncture can specifically regulate metallothionein-2（MT-2）gene expression through Serial Analysis of Gene Expression（SAGE）.However,the relationship between acupuncture treatment and MT-2 protein expression during asthma is still unclear,and the effect of MT-2 on the phosphorylation changes of airway smooth muscle cells（ASMCs）is also unclear.This article examines the changes of MT-2 protein after acupuncture treatment of asthma models to clarify the relationship between acupuncture and MT-2 protein expression in lung tissue.Functional experiments were used to clarify the effect of MT-2 on ASMC contraction and relaxation.High-throughput technology and bioinformatics analysis were used to clarify the phosphorylation mechanism of MT-2on ASMC contraction and relaxation.Methods:Four groups of rat models were established:a normal control（NC）group,an asthmatic model（AS）group,an asthmatic model group treated with acupuncture（AA）,and a group of normal rats treated with acupuncture（NA）.The AA group and the NA group were treated with acupuncture（GV14,bilateral BL12 and bilateral BL13）for 30 mins.Acupuncture once every other day,a total of 7 times.Separate rat lung tissue and use liquid nitrogen grinding method to extract total tissue protein.The Western blot method was used to measure the expression of MT-2 protein in the four groups and perform quantitative analysis.Constructed p ET-32a prokaryotic expression vector.The MT-2 protein was purified by nickel ion affinity chromatography.The primary rat airway smooth muscle cells（ASMCs）were cultured,and the cell purity was identified by cellular immunofluorescence withα-actin,a specific marker of smooth muscle.The purified ASMCs were incubated with 100 ng/ml,200 ng/ml,400 ng/ml MT-2 protein for 10minutes,and 1.5μM Terbutaline（TB）,a conventionalβ₂receptor agonist,was used as a positive control.Polarization microscope was used to determine the effect of MT-2protein on the contraction and relaxation of ASMCs.On the basis of the effect of MT-2 on relaxing rat ASMCs,using a Phospho Explorer antibody microarray（PEX100）,to analyze the phosphorylation changes of downstream proteins.Western blot was used to measure the phosphorylation changes of Akt1 and Ca MK2β.Use the PANTHER,DAVID and STRING for bioinformatics analysis.Results:1.Acupuncture regulates the expression of metallothionein-2 in lung tissue of asthmatic ratsCompared with the normal control group,the MT-2 protein expression in asthmatic rats was significantly reduced（P<0.05 vs NC,n=5）.The MT-2 protein expression in the AA group was significantly increased after acupuncture treatment（P<0.05 vs AS,n=5）.Compared with the normal control group,there was no statistically significant difference in MT-2 protein expression after acupuncture in normal rats.2.Metallothionein-2 recombinant protein has been obtained successfullyUsing nickel ion affinity chromatography to obtain 150μl of 2.4 mg/ml rat MT-2protein,a total of 360μg protein.3.Metallothionein-2 relaxs airway smooth muscle cellsRat ASMCs were identified by immunofluorescence using antibodies againstα-actin,a specific marker of ASMCs.The results showed that more than 90%of the cells are rat ASMCs,which can be used for ASMCs contraction and relaxation assays.Polarization microscope was used to measure the effect of MT-2 on the ASMC relaxation.The results showed that 1.5μM TB can relax ASMCs by 19.5%±3.9%（P<0.05 vs control）.In addition,100,200 or 400 ng/ml MT-2 can separately relax ASMCs by 12.8%±4.5%,16.1%±3.7%,and 18.4%±2.6%（P<0.05 vs control）.4.Metallothionein-2 causes changes in protein phosphorylation in airway smooth muscle cellsA Phospho Explorer antibody microarray was used to detect the phosphorylation changes in the downstream pathways of rat ASMCs induced by MT-2,and the results showed that the phosphorylation of 51 proteins has changed.The phosphorylation level of 14 proteins（27.5%）was up-regulated（P<0.05）,and the phosphorylation level of 37 proteins（72.5%）was down-regulated（P<0.05）.5.Verification of the phosphorylation changes caused by metallothionein-2Western blot found that the phosphorylation of Akt1 Thr450 was significantly increased by 4.07±0.13 times（P<0.05 vs control,n=3）,and the phosphorylation of Ca MK2βThr287 was significantly increased by 2.35±0.34 times（P<0.05 vs control,n=3）.The expression levels of phosphorylated Akt1 and Ca MK2βwere consistent in the Phospho Explorer antibody microarray.6.Bioinformatic analysis of metallothionein-2-induced protein phosphorylation The PANTHER classification has identified 51 phosphorylated proteins into 11functional groups,including transferase,calcium-binding protein,transcription factor,nucleic acid binding,enzyme modulator,transfer/carrier protein,hydrolase,receptor,defense immunity protein,cytoskeletal protein,signaling molecule.Akt1 exists in 3categories,including transfer/carrier protein,transferase and calcium binding protein.DAVID divided 51 phosphorylated proteins into 8 groups,including protein phosphorylation,regulation of GTPase activity,TCR signaling,cell proliferation,transcriptional regulation of RNA polymerase II promoter,angiogenesis,cell division and apoptosis.Akt1 was involved in the process of protein phosphorylation;Ca MK2βwas involved in protein phosphorylation and the regulation of GTPase activity.STRING showed that Akt1 is directly connected with 5 other proteins.Conclusion:Acupuncture can significantly increase the expression of MT-2 protein in the lung tissue of asthmatic rats,and achieve airway smooth muscle relaxation by regulating a series of protein phosphorylation including Akt1,Ca MK2β;thus suggesting that MT-2may play a role in the treatment of asthma by acupuncture Important role.