Yuan Lu1, Jian-Ya Xu2, Xiao-Hua Zhang3, Xia Zhao4. 1. Pediatric Institution of Nanjing University of Chinese Medicine, Nanjing 210023, China; Jiangsu Key Laboratory of Pediatric Respiratory Disease, Nanjing 210023, China. Electronic address: heiheidoctor@163.com. 2. Pediatric Institution of Nanjing University of Chinese Medicine, Nanjing 210023, China; Jiangsu Key Laboratory of Pediatric Respiratory Disease, Nanjing 210023, China. Electronic address: 710735592@qq.com. 3. Pediatric Institution of Nanjing University of Chinese Medicine, Nanjing 210023, China; Jiangsu Key Laboratory of Pediatric Respiratory Disease, Nanjing 210023, China. Electronic address: 2461969297@qq.com. 4. Pediatric Institution of Nanjing University of Chinese Medicine, Nanjing 210023, China; Jiangsu Key Laboratory of Pediatric Respiratory Disease, Nanjing 210023, China. Electronic address: zhaoxiahy@126.com.
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE: In recent years, asthma has increased dramatically in prevalence with a considerable economic burden all over the world. Long-term remission should be regarded as the promising and meaningful therapeutic goal in asthma management. However, the precise definition criteria and rational therapies for asthma remission have not been well-established. In academia, there is a consensus that even in those who develop asymptomatic remission of asthma, persistent airway inflammation is ubiquitous. Gubenfangxiao decoction (GBFXD) has been widely used in treating asthma remission stage for decades in the Jiangsu Province Hospital of Chinese Medicine, China. We previously demonstrated that GBFXD could downregulate the asthma susceptibility gene ORMDL3, a trigger of Endoplasmic reticulum (ER) stress and unfolded protein response (UPR). AIM THIS STUDY: To investigate the involvement of ER stress and UPR in the anti-inflammatory effects of GBFXD in Respiratory Syncytial Virus (RSV)-OVA-induced asthma remission mice. MATERIALS AND METHODS: Mice were orally administered GBFXD at three doses for 30 days after an RSV-OVA challenge. The levels of inflammation mediators in serum were measured using a Luminex assay and the amount of IFN-γ in lung homogenates was detected using ELISA. The splenic CD4+ and CD8+ T lymphocytes were counted using flow cytometric analysis. The mRNA and protein levels of asthma susceptibility gene ORMDL3, ER stress markers (BIP, CHOP), and three canonical UPR branches (PERK-eIF2a-ATF4, IRE1α-XBP1/IRE1α-JNK-AP1 and ATF6-SERCA2b signal pathways) were detected using real-time RT-PCR and western blot. RESULTS: Histopathological analysis showed that the model group mice still exhibited a sustained airway inflammation even after suspending the OVA-challenge and RSV infections for 30 days. H&E staining results indicated that GBFXD could attenuate sustained airway inflammation. Decreased serum CXCL1 level and increased IFN-γ level in lung homogenate were observed after GBFXD treatment. Reductions in the number of splenic CD4+/CD8+ T lymphocytes were found after DEX treatment. We further confirmed the previous finding that GBFXD could downregulate the expression of ORMDL3. As a result of suppressed UPR, decreased ER stress markers and inhibited UPR branches (PERK and IRE1α signal pathway) were also observed through the significant reduction of signature mRNA and protein expressions after GBFXD treatment. CONCLUSION: GBFXD can significantly attenuate RSV-OVA-induced persistent airway inflammation in murine asthma remission model. These effects may be mediated, at least partially, by inhibiting the activation of ER stress responses.
ETHNOPHARMACOLOGICAL RELEVANCE: In recent years, asthma has increased dramatically in prevalence with a considerable economic burden all over the world. Long-term remission should be regarded as the promising and meaningful therapeutic goal in asthma management. However, the precise definition criteria and rational therapies for asthma remission have not been well-established. In academia, there is a consensus that even in those who develop asymptomatic remission of asthma, persistent airway inflammation is ubiquitous. Gubenfangxiao decoction (GBFXD) has been widely used in treating asthma remission stage for decades in the Jiangsu Province Hospital of Chinese Medicine, China. We previously demonstrated that GBFXD could downregulate the asthma susceptibility gene ORMDL3, a trigger of Endoplasmic reticulum (ER) stress and unfolded protein response (UPR). AIM THIS STUDY: To investigate the involvement of ER stress and UPR in the anti-inflammatory effects of GBFXD in Respiratory Syncytial Virus (RSV)-OVA-induced asthma remission mice. MATERIALS AND METHODS:Mice were orally administered GBFXD at three doses for 30 days after an RSV-OVA challenge. The levels of inflammation mediators in serum were measured using a Luminex assay and the amount of IFN-γ in lung homogenates was detected using ELISA. The splenic CD4+ and CD8+ T lymphocytes were counted using flow cytometric analysis. The mRNA and protein levels of asthma susceptibility gene ORMDL3, ER stress markers (BIP, CHOP), and three canonical UPR branches (PERK-eIF2a-ATF4, IRE1α-XBP1/IRE1α-JNK-AP1 and ATF6-SERCA2b signal pathways) were detected using real-time RT-PCR and western blot. RESULTS: Histopathological analysis showed that the model group mice still exhibited a sustained airway inflammation even after suspending the OVA-challenge and RSV infections for 30 days. H&E staining results indicated that GBFXD could attenuate sustained airway inflammation. Decreased serum CXCL1 level and increased IFN-γ level in lung homogenate were observed after GBFXD treatment. Reductions in the number of splenic CD4+/CD8+ T lymphocytes were found after DEX treatment. We further confirmed the previous finding that GBFXD could downregulate the expression of ORMDL3. As a result of suppressed UPR, decreased ER stress markers and inhibited UPR branches (PERK and IRE1α signal pathway) were also observed through the significant reduction of signature mRNA and protein expressions after GBFXD treatment. CONCLUSION: GBFXD can significantly attenuate RSV-OVA-induced persistent airway inflammation in murineasthma remission model. These effects may be mediated, at least partially, by inhibiting the activation of ER stress responses.