[Effect of moxibustion of acupoints for both lung and intestine disorders on lung inflammation and intestinal short-chain fatty acids in asthmatic model rats].

OBJECTIVE: To explore the mechanism of moxibustion in the treatment of asthmatic inflammation from the point of short-chain fatty acids (SCFAs) in rats with asthma. METHODS: A total of 48 SD rats (half male and half female) were randomly divided into 4 groups： normal, model, lung treatment and joint-treatment of lung and intestine (joint-treatment), with 12 rats in each group. The asthma model was made by subcutaneous (bilateral back and inguinal regions) and intraperitoneal injection of mixture solution of ovalbumin and aluminium hydroxide gel (on day 1 and 8) and followed by inhalation of atomized 1% ovalbumin (20 min from day 15, once daily for one week). Moxibustion was applied to bilateral "Feishu" (BL13) for rats of the lung treatment group or bilateral "Feishu" (BL13) and "Tianshu" (ST25) for rats of the joint treatment group. One hour after the intervention, the rats in the later three groups were separately given atomized 1% ovalbumin solution inhalation for 20 min. The treatment was conducted for 30 min, once daily for 14 consecutive days. At the end of the intervention, the percentage of inflammatory cells in blood was detected by biochemical method and histopathological changes of the lung were observed after H.E. staining. The inflammatory cells in the bronchoalveolar lavage fluid (BALF) were counted after Wright-Giemsa staining. The mRNA expressions of interleukin (IL)-4, IL-5, IL-13, IL-17, IL-33, leukotriene (LT), thymic stromal lymphopoietin (TSLP) and prostaglandin D2 (PGD2) were detected by real-time PCR, and the contents of SCFAs in rats' feces were detected by gas chromatography-mass spectrometry. RESULTS: Relevant to the normal group, the model group had an obvious increase in the percentages of neutrophils, lymphocytes and eosinophils in the blood, the percentages of neutrophils and eosinophils in the BALF, and in the expression levels of PGD2, TSLP, LT, IL-4, IL-5, IL-13, IL-17 and IL-33 mRNAs in the lung tissues (P<0.01, P<0.05), and a marked decrease in the contents of acetic acid, propionic acid, isobutyric acid, butyric acid and valeric acid in feces (P<0.05, P<0.01). After the treatment, the percentages of neutrophils and lymphocytes in the peripheral blood, eosinophils in the BALF, and the expression levels of PGD2, TSLP, LT, IL-4, IL-17, IL-33 mRNAs in the lung tissues in both the lung treatment and joint treatment groups, as well as neutrophils of BALF, and expression of IL-5 and IL-13 mRNAs in the joint treatment group were significantly down-regulated (P<0.01, P<0.05), while the contents of acetic acid, propionic acid and valerate in the lung treatment group, and acetic acid, propionic acid, isobutyric acid, butyric acid and valeric acid in the joint treatment group were all strikingly increased (P<0.05, P<0.01). The effect of the joint treatment was superior to that of lung treatment in down-regulating the expressions of LT and IL-5 mRNAs (P<0.05, P<0.01) and up-requlating the content of propionic acid (P<0.05). Results of H.E. staining showed thickened alveolar wall, infiltration of a large number of inflammatory cells and interstitial fibrous tissue hyperplasia around the bronchus and scattered arrangement of cells of the lung tissue in the model group, which was relatively milder in both lung treatment and joint treatment groups, particularly the later. CONCLUSION: Joint treatment of asthma from the lung and intestine can better regulate the contents of intestinal SCFAs and alleviate the inflammatory response of asthmatic model rats, thus, intestinal SCFAs may be involved in the process of moxibustion in improving inflammatory response.

[Effects of moxibustion at "Tianshu"（ST25） and "Shangjuxu" （ST37） on colonic metabolites and inflammatory factors in rats with Crohn's disease].

OBJECTIVE: To observe the effects of moxibustion at "Tianshu"（ST25） and "Shangjuxu"（ST37） on the colonic metabolites and inflammatory factors in rats with Crohn's disease（CD）, so as to explore the mechanisms of moxibustion in protecting colon of CD rats based on metabolomics. METHODS: Twelve rats were first randomly selected from 36 male SD rats as a normal group（NG）. The CD model was induced by 2, 4, 6 trinitrobenzene sulfonic acid（TNBS） enema on the rest 24 rats. After successful modeling, rats were randomly divided into model（TNBS） and moxibustion（TNBS+MOX） groups（n=10 rats/group）. Moxibustion was applied at bilateral ST25 and ST37 for 30 min, once daily for 7 consecutive days in the TNBS+MOX group, while rats in the NG and TNBS groups did not receive any interventions. Body weight of rats was recorded and disease activity index（DAI） was assessed during the experiment. After interventions, HE staining was performed to observe pathological damage of colon. Serum levels of inflammatory factors were measured by ELISA. NMR hydrogen spectroscopy was used to detect colonic metabolites of each group, and orthogonal partial least squares discriminant analysis（OPLS-DA） was used to screen differential colonic metabolites between groups, followed by pathway analysis using MetaboAnalyst 5.0 platform. RESULTS: After modeling, compared with the NG group, the body weight of the rats in the TNBS group was significantly decreased（P<0.05）, the DAI score was increased （P<0.05）, the colon had obvious inflammatory damage and the pathological injury index was increased（P<0.05）, and levels of serum tumor necrosis factor-α（TNF-α）, interleukin（IL）-1ß and interferon-γ（IFN-γ） were significantly increased（P<0.05）. After moxibustion intervention, compared with the TNBS group, the body weight was significantly increased（P<0.05）, while the levels of serum TNF-α, IL-1ß, IFN-γ, and DAI score of the rats in the TNBS+MOX group were significantly decreased（P<0.05）, with alleviated colonic inflammatory injury detected by HE staining. Compared with the NG group, the relative expressions of colonic hypoxanthine, betaine, creatine, inositol, taurine, uracil, and methanol of the TNBS group were decreased（P<0.05）, while the relative expressions of histidine, leucine, proline, lysine, isoleucine, phenylalanine, tyrosine, propionic acid, and valine were increased（P<0.05） in the TNBS group, among which, relative expressions of hypoxanthine, leucine, lysine, isoleucine, betaine, tyrosine, and taurine were reversed in the TNBS+MOX group relevant to the TNBS group, mainly involving phenylalanine, tyrosine and tryptophan biosynthesis, and taurine and subtaurine metabolism pathway. CONCLUSION: The mechanism of moxibustion at ST25 and ST37 for CD may be related to improving colon metabolic disorder state by regulating multiple metabolic metabolites and metabolic pathways, and reducing the level of inflammatory factors, so as to maintain intestinal immune homeostasis.

Retinoid X receptor agonists alleviate fibroblast activation and post-infarction cardiac remodeling via inhibition of TGF-ß1/Smad pathway.

Retinoid X receptor (RXR), particularly RXRα, has been implicated in cardiovascular diseases. However, the functional role of RXR activation in myocardial infarction (MI) remains unclear. This study aimed to determine the effects of RXR agonists on MI and to dissect the underlying mechanisms. Sprague-Dawley (SD) rats were subjected to MI and then treated (once daily for 4 weeks) with either RXR agonist bexarotene (10 or 30 mg/kg body weight) or vehicle. Heart function was determined using echocardiography and cardiac hemodynamic measurements. Four weeks post MI, myocardial tissues were collected to evaluate cardiac remodeling. Primary cardiac fibroblasts (CFs) were treated with or without RXR ligand 9-cis-RA followed by stimulation with TGF-ß1. Immunoblot, immunofluorescence, and co-immunoprecipitation were performed to elucidate the regulatory role of RXR agonists in TGF-ß1/Smad signaling. In vivo treatment with Bexarotene moderately affects systemic inflammation and apoptosis and ameliorated left ventricular dysfunction after MI in rat model. In contrast, bexarotene significantly inhibited post-MI myocardial fibrosis. Immunoblot analysis of heart tissue homogenates from MI rats revealed that bexarotene regulated the activation of the TGF-ß1/Smad signaling pathway. In vitro, 9-cis-RA inhibited the TGF-ß1-induced proliferation and collagen production of CFs. Importantly, upon activation by 9-cis-RA, RXRα interacted with p-Smad2 in cytoplasm, inhibiting the TGF-ß1-induced nuclear translocation of p-Smad2, thereby negatively regulating TGF-ß1/Smad signaling and attenuating the fibrotic response of CFs. These findings suggest that RXR agonists ameliorate post-infarction myocardial fibrosis, maladaptive remodeling, and heart dysfunction via attenuation of fibrotic response in CFs through inhibition of the TGF-ß1/Smad pathway activation.

[Effect of "joint treatment of lung and intestine" with moxibustion on lung function and airway inflammation in asthmatic rats].

OBJECTIVE: To investigate the role of moxibustion of "Feishu" (BL13)，"Tianshu" (ST25) for asthma by simultaneously treating lung and intestine (i.e., treating both lung and intestine at the same time) in asthmatic rats. METHODS: A total of 48 SD rats were randomly divided into normal, model, lung treatment and joint-treatment of lung and intestine (joint-treatment) groups, with 12 rats in each. The asthma model was established by subcutaneous (bilateral back and inguinal regions) and intraperitoneal injection of mixture solution of albumin and Aluminium Hydroxide gel (on day 1st, and 9th) and followed by inhalation of atomized 1% ovalbumin (on day 15th, 20 min each time, once daily for 1 week). Moxibustion was applied to bilateral BL13 for rats of the lung treatment group or bilateral BL13 and ST25 for rats of the joint-treatment group. One hour after the intervention, the rats in the later three groups were separately given nebulized 1% ovalbumin solution inhalation for 20 min. The treatments were conducted once daily for 14 consecutive days. After intervention, the lung functions including the forced expiratory flow 25% (FEF 25%), maximal mid-expiratory flow (MMEF), dynamic lung compliance (Cdyn), forced expiratory volume/ forced vital capacity (FEV/FVC), peak expiratory flow (PEF), and lung resistance (RL) were measured by using a small animal lung function detector, and pathological changes and collagen deposition in the lung tissues were observed by H.E. and Masson staining, separately. The levels of interleukin (IL)-17, IL-4, IL-13, IL-33, IL-5, leukotriene (LT) and thymic stromal lymphocyte (TSLP) in the lung tissue were measured by ELISA. RESULTS: Compared with the normal group, the FEF 25%, MMEF， Cdyn， FEV/FVC and PEF were significantly decreased (P<0.05, P<0.01)， and the pulmonary RL, collagen deposition, and contents of IL-17, IL-4, IL-13, IL-33, IL-5, TSLP and LT were notably increased (P<0.05, P<0.01) in the model group. After intervention, the MMEF and Cdyn in the lung treatment group, PEF, MMEF, Cdyn, FEV/FVC, FEF 25% in the joint-treatment group, were markedly increased (P<0.05, P<0.01), whereas the collagen deposition, IL-17, IL-4 and TSLP in both the lung treatment and joint-treatment groups, RL, IL-13, IL-33, IL-5 and LT in the joint-treatment group were considerably down-regulated (P<0.05, P<0.01). The effects of the joint treatment were apparently superior to those of lung treatment in down-regulating the contents of TSLP and LT (P<0.05, P<0.01). H.E. staining showed thickened alveolar wall, infiltration of a large number of inflammatory cells in the bronchus of the model group, which was relatively milder in the joint-treatment group. CONCLUSION: "Joint treatment of lung and intestine" with moxibustion is superior to "lung treatment" alone in ameliorating the lung function and mitigating airway inflammation in rats with asthma.

[Effect of moxibustion on respiratory function and cutaneous histamine and neuropeptide contents of "Feishu" (BL13) in asthmatic rats].

OBJECTIVE: To observe the effect of moxibustion on respiratory function and contents of histamine and neuropeptides in skin tissue of "Feishu" (BL13) in asthmatic rats, so as to explore its mechanisms underlying improvement of asthma. METHODS: Thirty-six SD rats were randomly divided into normal control, model and moxibustion groups, with 12 rats in each group. The asthma model was established by subcutaneous injection (at the back and groin, 0.5 mL) and i．p. injection (1 mL)of mixture solution of ovalbumin (OVA), Aluminium Hydroxide gel and 0.9% sodium chloride solution, and repeated nasal drip of 1% OVA. Moxibustion was applied to bilateral BL13 for 15 min, once daily for 14 days. The inspiratory and expiratory resistance, and pulmonary ventilation compliance were detected by using a small animal pulmonary function tester under anesthesia. The contents of histamine, vasoactive intestinal peptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP) in the local skin tissue of the left BL13 were assayed by using enzyme-linked immunosorbent assay (ELISA). RESULTS: After modeling, the inspiratory and expiratory resistance were significantly increased (P<0.01), and the lung ventilation compliance was significantly decreased in the model group relevant to the normal control group (P<0.01). Outcomes of ELISA showed that the content of VIP was significantly lower (P<0.01), and those of histamine, SP and CGRP in the skin of left BL13 were significantly increased in the model group relevant to the normal control group (P<0.01). Following the intervention, the inspiratory and expiratory resis-tance, cutaneous histamine, SP and CGRP contents were significantly decreased (P<0.01), while the lung ventilation compliance and cutaneous VIP level were significantly increased in the moxibustion group in contrast to the model group (P<0.01, P<0.05). CONCLUSION: Moxibustion of BL13 can improve pulmonary function in asthma rats, which may be related to changes of levels of histamine and neuropeptides as VIP, SP and CGRP in the local skin tissues of BL13.