Dong-Dong Zhou1, Jun Ran2, Cong-Cong Li1, Jin Lu1, Qing-Yi Zhao1, Xiao-Yan Liu1, Yu-Dong Xu1, Yu Wang1, Yong-Qing Yang3, Lei-Miao Yin4. 1. Laboratory of Molecular Biology, Shanghai Research Institute of Acupuncture and Meridian, Shanghai University of Traditional Chinese Medicine, Shanghai, 200030, China. 2. Laboratory of Molecular Biology, Shanghai Research Institute of Acupuncture and Meridian, Shanghai University of Traditional Chinese Medicine, Shanghai, 200030, China; Chongqing Hospital of Traditional Chinese Medicine, Chongqing, 400021, China. 3. Laboratory of Molecular Biology, Shanghai Research Institute of Acupuncture and Meridian, Shanghai University of Traditional Chinese Medicine, Shanghai, 200030, China. Electronic address: yyq@shutcm.edu.cn. 4. Laboratory of Molecular Biology, Shanghai Research Institute of Acupuncture and Meridian, Shanghai University of Traditional Chinese Medicine, Shanghai, 200030, China; Shanghai Innovation Center of Traditional Chinese Medicine Health Service, Shanghai, 201203, China. Electronic address: collegeylm@shutcm.edu.cn.
Abstract
BACKGROUND: Acupuncture has long been used for asthma treatment but the underlying mechanism remains unclear. Previous study showed that metallothionein-2 (MT-2) was significantly decreased in asthmatic lung tissue. However, the relationship between acupuncture treatment and MT-2 expression during asthma is still unknown, and the detailed effect analysis of MT-2 on phosphorylation in airway smooth muscle cells (ASMCs) is also unclear. METHODS: The acupuncture effect on pulmonary resistance (RL) was investigated in a rat model of asthma, and the mRNA and protein levels of MT-2 in lung tissue were detected. Primary ASMCs were isolated and treated with MT-2 recombinant protein to study the MT-2 effects on ASMC relaxation. A Phospho Explorer antibody microarray was applied to detect protein phosphorylation changes associated with MT-2-induced ASMC relaxation. Bioinformatic analysis were performed with PANTHER database, DAVID and STRING. Phosphorylation changes in key proteins were confirmed by Western blot. RESULTS: Acupuncture significantly reduced RL at 2-5 min (P < 0.05 vs asthma) in asthmatic rats. Acupuncture continued to increase MT-2 mRNA expression in lung tissue for up to 14 days (P < 0.05 vs asthma). The MT-2 protein expression was significantly decreased in the asthmatic rats (P < 0.05 vs control), while MT-2 protein expression was significantly increased in the asthmatic model group treated with acupuncture (P < 0.05 vs asthma). Primary ASMCs were successfully isolated and recombinant MT-2 protein (100, 200, 400 ng/ml) significantly relaxed ASMCs (P < 0.05 vs control). MT-2 induced phosphorylation changes in 51 proteins. Phosphorylation of 14 proteins were upregulated while 37 proteins were downregulated. PANTHER classification revealed eleven functional groups, and the phosphorylated proteins were identified as transferases (27.8 %), calcium-binding proteins (11.1 %), etc. DAVID functional classification showed that the phosphorylated proteins could be attributed to eight functions, including protein phosphorylation and regulation of GTPase activity. STRING protein-protein interaction network analysis showed that Akt1 was one of the most important hubs for the phosphorylated proteins. The phosphorylation changes of Akt1 and CaMK2β were consistent in both the Phospho Explorer antibody microarray and Western blot. CONCLUSION: Acupuncture can significantly ameliorate RL, and the MT-2 mRNA and protein levels in lung tissue are increased during treatment. MT-2 significantly relaxes ASMCs and induces a series of protein phosphorylation. These phosphorylation changes, including Akt1 and CaMK2β, may play important roles in the therapeutic effects of acupuncture on asthma.
BACKGROUND: Acupuncture has long been used for asthma treatment but the underlying mechanism remains unclear. Previous study showed that metallothionein-2 (MT-2) was significantly decreased in asthmatic lung tissue. However, the relationship between acupuncture treatment and MT-2 expression during asthma is still unknown, and the detailed effect analysis of MT-2 on phosphorylation in airway smooth muscle cells (ASMCs) is also unclear. METHODS: The acupuncture effect on pulmonary resistance (RL) was investigated in a rat model of asthma, and the mRNA and protein levels of MT-2 in lung tissue were detected. Primary ASMCs were isolated and treated with MT-2 recombinant protein to study the MT-2 effects on ASMC relaxation. A Phospho Explorer antibody microarray was applied to detect protein phosphorylation changes associated with MT-2-induced ASMC relaxation. Bioinformatic analysis were performed with PANTHER database, DAVID and STRING. Phosphorylation changes in key proteins were confirmed by Western blot. RESULTS: Acupuncture significantly reduced RL at 2-5 min (P < 0.05 vs asthma) in asthmatic rats. Acupuncture continued to increase MT-2 mRNA expression in lung tissue for up to 14 days (P < 0.05 vs asthma). The MT-2 protein expression was significantly decreased in the asthmatic rats (P < 0.05 vs control), while MT-2 protein expression was significantly increased in the asthmatic model group treated with acupuncture (P < 0.05 vs asthma). Primary ASMCs were successfully isolated and recombinant MT-2 protein (100, 200, 400 ng/ml) significantly relaxed ASMCs (P < 0.05 vs control). MT-2 induced phosphorylation changes in 51 proteins. Phosphorylation of 14 proteins were upregulated while 37 proteins were downregulated. PANTHER classification revealed eleven functional groups, and the phosphorylated proteins were identified as transferases (27.8 %), calcium-binding proteins (11.1 %), etc. DAVID functional classification showed that the phosphorylated proteins could be attributed to eight functions, including protein phosphorylation and regulation of GTPase activity. STRING protein-protein interaction network analysis showed that Akt1 was one of the most important hubs for the phosphorylated proteins. The phosphorylation changes of Akt1 and CaMK2β were consistent in both the Phospho Explorer antibody microarray and Western blot. CONCLUSION: Acupuncture can significantly ameliorate RL, and the MT-2 mRNA and protein levels in lung tissue are increased during treatment. MT-2 significantly relaxes ASMCs and induces a series of protein phosphorylation. These phosphorylation changes, including Akt1 and CaMK2β, may play important roles in the therapeutic effects of acupuncture on asthma.