Cigarette smoke extract-induced inflammatory response via inhibition of the TFEB-mediated autophagy in NR8383 cells.

Objective: Chronic pulmonary inflammation caused by long-term smoking is the core pathology of COPD. Alveolar macrophages (AMs) are involved in the pulmonary inflammation of COPD. The accumulation of damaged materials caused by impaired autophagy triggers inflammatory response in macrophages. As a key transcription regulator, transcription factor EB (TFEB) activates the transcription of target genes related autophagy and lysosome by binding to promoters, whereas it is unclarified for the relationship between inflammatory response induced by cigarette smoke extract (CSE) and TFEB-mediated autophagy. Thus, we investigated the role of TFEB-mediated autophagy in inflammatory response induced by CSE in NR8383 cells, and to explore its potential mechanism. Methods: Based on cell viability and autophagy, cells treated with 20% concentration of CSE for 24 h were selected for further studies. Cells were divided into control group, chloroquine (CQ, the autophagy inhibitor) group, CSE group, CSE + rapamycin (the autophagy inducer) group and CSE + fisetin (the TFEB inducer) group. The levels of tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and IL-6 in supernatant were detected by ELISA kits. The protein expressions were tested by western blot. The intensity of fluorescence of Lysosome-associated membrane protein 1 (LAMP1) and TFEB was detected by immunofluorescence. Lyso-Tracker Red staining was applied to detect the lysosome environment. Results: CSE inhibited the cell viability, increased the contents of TNF-α, IL-1ß, IL-6, the ratio of LC3II/I, and the level of P62 protein. Besides, CSE decreased the fluorescence intensity of LAMP1 protein and Lyso-Tracker Red staining, as well as the ratio of nucleus/cytosol of TFEB protein. Activating autophagy with rapamycin alleviated CSE-induced inflammatory response. The activation of TFEB via fisetin alleviated CSE-induced autophagy impairment and lysosomal dysfunction, thus alleviated inflammatory response in NR8383 cells. Conclusion: CSE-induced inflammatory response in NR8383 cells, which may be related to the inhibition of TFEB-mediated autophagy.

The Pretreatment of Xiaoqinglong Decoction Alleviates Inflammation and Oxidative Damage and Up-Regulates Angiotensin-Converting Enzyme 2 in Lipopolysaccharide-Induced Septic Acute Lung Injury Rats.

Xiaoqinglong decoction (XQLD), a classic prescription of Traditional Chinese Medicine, has already been used clinically to cure acute lung injury (ALI), but its mechanism remains unclear. This subject aimed to explore the preventive role of XQLD in septic ALI rats besides its effects on angiotensin-converting enzyme (ACE)2 and its downstream factors. After, respectively, administrated with different concentrations of XQLD (6.25 g/kg/d, 12.5 g/kg/d, 25 g/kg/d) for 5 days and dexamethasone (DEX, 1 mg/kg) for 0.5 h, the rat models of ALI were established by intraperitoneal injection of lipopolysaccharide (LPS, 5 mg/kg) for 24 h. All rats were evaluated by lung function test, arterial blood gas analysis, morphological observation, lung wet/dry (W/D) ratio, and the lung injury score. The levels of malonaldehyde (MDA), superoxide dismutase (SOD), interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and angiotensin (Ang) (1-7) in the lung were measured through biochemical and ELISA kits. The expressions of angiotensin-converting enzyme (ACE)2, mitochondrial assembly receptor (MasR), and nuclear factor (NF)-κB in lung tissue were detected by qRT-PCR and western blotting. Positive reaction cells of MasR were observed by immunohistochemistry. The results show that XQLD significantly ameliorated septic lung injury including edema and hemorrhage, as well as improved pulmonary function and arterial blood gas. Furthermore, XQLD markedly decreased the levels of IL-1ß, TNF-α, MDA, and NF-κB while increased the levels of SOD, Ang (1-7), ACE2, and MasR in septic ALI rats. Pearson correlation showed that the expressions of ACE2 were inversely related to IL-1ß, TNF-α, MDA, and NF-κB and positively correlated with SOD contents. Our data indicated that XQLD pretreatment alleviated inflammation and oxidative damage in septic ALI rats, which might be related to the up-regulation of ACE2-Ang (1-7)-MasR axis and inhibition of the NF-κB pathway.

[Electroacupuncture preconditioning improves pulmonary function via inhibiting inflammatory response and up-regulating expression of ACE2 and Ang (1-7) in lipopolysaccharide-induced acute lung injury rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Zusanli"(ST36) pretreatment on lung functions, inflammatory response, and levels of angiotensin-converting enzyme 2 (ACE2) and angiotensin (1-7) ï¼»Ang (1-7)ï¼½ in rats with sepsis-induced acute lung injury (ALI), so as to explore its mechanisms underlying improvement of ALI. METHODS: Thirty male SD rats were randomly divided into normal, model and EA groups (n=10 in each group). The sepsis-related ALI model was established by intraperitoneal injection of lipopolysaccharide (LPS, 5 mg/kg). Rats of the EA group received EA (4 Hz/20 Hz, 1-3 mA) stimulation at bilateral ST36 for 30 min, once each day, for 7 days before modeling. The lung functions including forced vital capacity (FVC), forced expiratory volume at 0.1 second (FEV0.1) and FEV0.3 were detected using a respiratory function detector for small animals at 3 h after modeling. The bronchoalveolar lavage fluid (BALF) was collected for assaying the contents of Ang (1-7), tumor necrosis factor-α (TNF-α) and interleukin-1 ß (IL-1ß) using ELISA. The lung wet/dry weight (W/D) ratio, FEV0.1/FVC, and FEV0.3/FVC were calculated. The histopathological changes of lung tissues were displayed by hematoxylin-eosin (H.E.) staining. The expression of ACE2 and mitochondrial assembly receptor (MasR) mRNAs and proteins in the lung tissue was detected by fluorescence quantitative real-time PCR and Western blot, separately. RESULTS: Following modeling, the levels of FVC, FEV0.1, FEV0.3, ratio of FEV0.1/FVC and FEV0.3/FVC, content of Ang (1-7) in the BALF, and the expression levels of ACE2 and MasR mRNAs and proteins in the lung tissue were significantly decreased (P<0.01), while the level of W/D ratio and TNF-α and IL-1ß contents in the BALF significantly increased (P<0.01) in the model group relevant to the normal group. In comparison with the model group, the levels of FVC, FEV0.1, FEV0.3, ratio of FEV0.1/FVC and FEV0.3/FVC, content of Ang (1-7) in the BALF, and expression levels of ACE2 and MasR mRNAs and proteins in the lung tissue were significantly increased (P<0.05, P<0.01), whereas the level of W/D ratio, and TNF-α and IL-1ß contents in the BALF were significantly decreased (P<0.05, P<0.01) in the EA group. H.E. staining showed pulmonary interstitial edema and alveolar septum thickening with severe inflammatory cell infiltration in the model group, which was relatively milder in the EA group. CONCLUSION: EA preconditioning at ST36 can improve pulmonary function in sepsis-related ALI rats, which may be related to its effects in inhibiting inflammatory response and up-regulating ACE2 and MasR expression and Ang (1-7) content in the lung tissue.

[Effect of electroacupuncture on pulmonary neuroendocrine cells and secretion of neuroactive substances in lung of COPD rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Zusanli" (ST36) and "Feishu" (BL13) on the activation and secretion of calcitonin gene-related peptide (CGRP) and 5-hydroxytryptamine (5-HT) of pulmonary neuroendocrine cells (PNECs) and inflammatory response in rats with chronic obstructive pulmonary disease (COPD), so as to explore its underlying mechanisms in treating COPD. METHODS: Male SD rats were randomly divided into normal control, COPD model and EA groups, with 7 rats in each group. The COPD model was established by forced inhale of cigarette smoke for 1 h in a self-made box (1 m×1 m×1 m in volume), twice daily for 12 weeks. EA (4 Hz/20 Hz, 1-3 mA) was applied at bilateral ST36 and BL13 acupoints for 30 min, once a day for 14 consecutive days. The pulmonary function including the forced vital capacity (FVC), forced expiratory volume at 0.1 second (FEV0.1), FEV0.3, FEV0.1/FVC and FEV0.3/FVC was detected using a lung function analyzer for small animals. The lung tissue was sampled for observing histopathological changes by using H.E. staining, for observing expression and distribution of PNECs by Grimelius silver staining, and for detecting the immunoactivity (integrated optical density) of CGRP and 5-HT by using immunohistochemistry. The contents of CGRP, 5-HT, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and transforming growth factor-ß1 (TGF-ß1) in the bronchoalveolar lavage fluid (BALF) and lung tissue were detected by ELISA, and the correlations between TNF-α and CGRP, IL-1ß and CGRP, TNF-α and 5-HT, and IL-1ß and 5-HT levels were analyzed. The mRNA and protein expression levels of nerve fiber markers of CGRP and purinergic receptor P2X ligand gated ion channel 3 (P2X3) which dominate PNECs in the lung tissue were detected by real-time fluorescence quantitative PCR and Western blot, respectively. RESULTS: Compared with the normal control group, the levels of FVC, FEV0.1, FEV0.3, and the ratios of FEV0.1/FVC and FEV0.3/FVC were significantly decreased (P<0.05, P<0.01), while the immunoactivity of PNECs, CGRP and 5-HT, the contents of CGRP, 5-HT, TNF-α, IL-1ß and TGF-ß1 in the BALF and lung tissue, and the expression levels of CGRP and P2X3 mRNAs and proteins in the lung tissue significantly increased in the COPD model group (P<0.01, P<0.05). Following EA intervention, both the increased and decreased levels of all the indexes mentioned above were reversed (P<0.05, P<0.01) except FEV0.3. H.E. staining showed severe deformed bronchial lumen with thickened wall and alveolar septum, and obvious inflammatory cell infiltration and reduced number of alveolar lumen fusion in the COPD model group, which was mild in the EA group. A positive correlation was found between TNF-α and CGRP, IL-1ß and CGRP, TNF-α and 5-HT,IL-1ß and 5-HT levels in both BALF and lung tissues (P<0.01). CONCLUSION: EA at ST36 and BL13 can improve lung function and reduce inflammatory response in COPD rats, which may be related to its function in inhibiting the activation of PNECs and release of neuroactive substances.

Hydrogen regulates the M1/M2 polarization of alveolar macrophages in a rat model of chronic obstructive pulmonary disease.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a respiratory disease with high morbidity and mortality worldwide, so far there is no ideal treatment method. Previous studies have shown that hydrogen (H2) is involved in the treatment of COPD as an antioxidant. In this study, the effect of H2 on M1/M2 polarization of alveolar macrophages in COPD rats was observed, and its anti-inflammatory mechanism was further elucidated. Methods: Twenty-four Sprague-Dawley rats were randomly divided into three groups including the control, COPD and H2 group. A rat model of COPD was established by cigarette exposure combined with lipopolysaccharide (LPS) induction. H2 therapy was administered 2 hours per day for 14 days. Lung function and pathology were assessed. The levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß1 and IL-10 in bronchoalveolar lavage fluid (BALF) and lung tissue were measured by enzyme-linked immunosorbent assay. The mRNA, protein expression and immunoreactivity of inducible nitric oxide synthase (iNOS) and arginase (Arg)-1 in lung were observed by quantitative real-time PCR, western blot and immunohistochemistry. Results: Compared with the control rats, there were a significant decline in lung function, a marked inflammatory infiltration and pulmonary parenchymal remodeling and the increases of IL-6, TNF-α and TGF-ß1 levels in BALF and lung tissue, but a lower expression of IL-10 in COPD rats. The iNOS mRNA and protein expression, as well as its optical density (OD), were increased significantly in lung tissue, while those of Arg-1 decreased significantly. H2 treatment improved the lung function and the parenchymal inflammation, reversed the increased levels of IL-6, TNF-α and TGF-ß1, and the lower IL-10. Meanwhile, H2 also down-regulated the expression of iNOS, but up-regulated expression of Arg-1 in lung tissue. Conclusion: H2 reduces inflammation in the lung of COPD, which may be related to its inhibition of M1 type polarization and activation of M2 type polarization of alveolar macrophage.

[Effect of electroacupuncture on the expression of autophagy related protein in lung tissue of rats with chronic obstructive pulmonary disease].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Zusanli" (ST36) and "Feishu" (BL13) on the expression of autophagy related proteins in the lung tissue of rats with chronic obstructive pulmonary disease (COPD), so as to explore the mechanism of EA underlying improvement of COPD. METHODS: Thirty male SD rats were randomly divided into normal, model and EA groups (n=10 in each group). The COPD model was established by intratracheal infusion of Lipopolysaccharide (LPS, 1 mg/kg) and exposure in cigarette smoke. EA was applied to bilateral ST36 and BL13 for 30 min, once every other day for 2 weeks. The pulmonary function (forced vital capacity ï¼»FVCï¼½, forced expiratory volume in 0.1 s and 0.3 s ï¼»FEV0.1, FEV0.3ï¼½, FEV0.1/FVC and FEV0.3/FVC) was detected by animal pulmonary function analysis system. Histopathological changes of the airway and lung were displayed by H.E. staining. Autophagosomes in the airway and lung tissues were observed by electron microscope. The expression of AMP activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), Unc-51 like autophagy activating kinase 1(ULK1), autophagy related protein ATG6(Beclin1)mRNAs in lung tissue were examined by quantitative real-time PCR. The expression of AMPK, mTOR, ULK1, Beclin1 and microtubule-associated protein 1 light chain 3 (LC3)proteins in lung tissue were examined by Western blot. The contents of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) in the broncho alveolar lavage fluid (BALF) were assayed by ELISA. RESULTS: Following modeling, the FVC, FEV0.1, FEV0.3, FEV0.1/FVC and FEV0.3/FVC levels were significantly decreased (P<0.01), the infiltration of inflammatory cells and the increase of autophagosomes were obvious in airway and lung tissue, the mRNA and protein expression of AMPK, ULK1, Beclin1 and the ratio of LC3Ⅱ/LC3Ⅰ were increased (P<0.01), while the mRNA and protein expression of mTOR were decreased (P<0.01), the contents of TNF-α and IL-6 in the BALF were increased in the model group compared with the normal group (P<0.01). After EA intervention, all the indexes mentioned above were completely reversed in the EA group relevant to the model group (P<0.01, P<0.05). CONCLUSION: EA at ST36 and BL13 can improve the lung function of COPD rats, which may be related to its effects in inhibiting the autophagy level and reducing the inflammation response in the lung.

[Electroacupuncture improves lung function by suppressing mucin-5AC mediated EGFR-p38MAPK signaling and inflammation reaction in chronic obstructive pulmonary disease rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Zusanli"(ST36) on the expression of epidermal growth factor receptor (EGFR), tumor necrosis factor α(TNF-α) transfer growth factor α(TGF-α), interleukin-8(IL-8), p38 mitogen-activated protein kinases (p38MAPK), mucin-5AC (MUC5AC) and other related factors in chronic obstructive pulmonary disease (COPD) rats, so as to reveal its underlying mechanisms in improving COPD. METHODS: A total of thirty male SD rats were randomly divided into normal control, model and EA groups, with 10 rats in each group. The COPD model was replicated using a combined method of tracheal infusion of lipopolysaccharide (LPS) and forced smoke-inhaling. EA (1-3 mA, 4 Hz/20 Hz) was applied to bilateral ST36 for 30 min, once daily for two consecutive weeks. The lung ventilation activities including the forced vital capacity (FVC) and forced expiratory volume (FEV) at 0.1 and 0.3 s (FEV0.1, FEV0.3) were detected. Histopathological changes of the middle lobe and bronchus of the right lung were observed after H.E. staining. The contents of TGF-α, TNF-α and IL-8 in the serum, bronchoalveolar lavage fluid (BALF) and superior lobe of the right lung were assayed by using ELISA, and the expression levels of EGFR, p38MAPK and MUC5AC proteins (inferior lobe of the left lung) and mRNAs (inferior lobe of the right lung) detected using Western blot, immunohistochemistry (strept avidin-biotin complex, SABC method) and real-time quantitative PCR, respectively. RESULTS: Compared with the normal group, the FVC, FEV0.1, FEV0.3, FEV0.1/FVC and FEV0.3/FVC levels were significantly decreased (P<0.01), while the contents of TNF-α, TGF-α and IL-8 in the serum, BALF and lung tissues, expression levels of EGFR, p38MAPK and MUC5AC mRNAs and proteins, and the immunoactivity of EGFR, p38MAPK and MUC5AC in the lung tissues were significantly increased in the model group (P<0.01). After EA intervention, the decreased levels of the FVC, FEV0.1, FEV0.3, FEV0.1/FVC and FEV0.3/FVC, and the increased levels of the abovementioned genes and proteins were all reversed in the EA group (P<0.01, P<0.05). After modeling, the bronchial walls were thickened, with enlarged alveolar cavities, fractured alveolar walls, obvious inflammatory cell infiltration, and rich mucus secretion in the lumen, which was relatively milder in the EA group. CONCLUSION: EA of ST36 can improve the ventilation function in COPD rats, which may be associated with its function in down-regulating the levels of TNF-α, TGF-α, IL-8, EGFR, p38MAPK and MUC5AC mRNAs and proteins in the lung tissues, inhibiting EGFR-p38MAPK signaling mediated expression of MUC5AC.

Electroacupuncture inhibits IL-17/IL-17R and post-receptor MAPK signaling pathways in a rat model of chronic obstructive pulmonary disease.

OBJECTIVE: Interleukin (IL)-17, as a T-helper 17 cell (Th17) cytokine, plays a key role in chronic obstructive pulmonary disease (COPD) pathophysiology including chronic inflammation and airway obstruction, which lead to decreased pulmonary function. The aim of this study was to investigate the effect of acupuncture on IL-17, its receptor (IL-17R) and the mitogen-activated protein kinase (MAPK) signaling pathway, in a rat model of COPD. METHODS: The COPD model was induced in Sprague Dawley rats by exposure to cigarette smoke for 12 weeks. The model rats were treated with electroacupuncture (EA) at BL13 and ST36. The lung function and histology of the rats were observed. IL-17, tumor necrosis factor (TNF)-α, and IL-10 were detected by enzyme-linked immunosorbent assay (ELISA) in bronchoalveolar lavage fluid (BALF) and in plasma. The leukocytes and macrophages in the BALF were counted. The expression levels of IL-17R were assayed in lung tissue by real-time polymerase chain reaction (PCR), western blotting, and immunohistochemistry. MAPK signaling pathway molecules including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK)1/2 and p38, and their phosphorylated forms, were observed in the lung by western blotting. RESULTS: Compared with the control group rats, lung function decreased and there was a severe inflammatory infiltration of the pulmonary parenchyma in the COPD rats. EA effectively improved lung function and alleviated the inflammatory infiltration in the lungs of COPD rats. EA also reversed the elevated total leukocyte and macrophage counts, the high levels of IL-17 and TNF-α, and the low IL-10 content in COPD rats. Meanwhile, EA downregulated the increased mRNA and protein expression of IL-17R, and significantly inhibited the elevated levels of phosphorylated JNK, ERK1/2, and p38 in the lungs of COPD rats. CONCLUSION: Our results suggest that the protective effects of acupuncture therapy on the lungs of COPD rats are likely related to inhibition of IL-17/IL-17R and the post-receptor MAPK signaling pathways.

[Effect of electroacupuncture on pulmonary function and M1 polarization of alveolar macrophages in rats with chronic obstructive pulmonary disease].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Zusanli" (ST36) and "Feishu" (BL13) on M1 polarization of alveolar macrophages (AM) in rats with chronic obstructive pulmonary disease(COPD), so as to explore its anti-inflammatory mechanism underlying improvement of COPD. METHODS: Forty SD rats were randomly divided into normal and normal＋EA, COPD model and COPD＋EA groups (n＝10 in each group). The COPD model was established by simple fumigation. EA (4 Hz/20 Hz, 1 to 2 mA) was applied to bilateral ST36 and BL13 for 30 min, once every other day for 2 weeks. The pulmonary function including the forced vital capacity (FVC), forced expiratory volume in 0.1 and 0.3 s (FEV0.1, FEV0.3, FEV0.1/FVC, and FEV0.3/FVC) was detected by using a small animal respiratory function detector. Histopathological changes of the lung were displayed by H．E. staining. The contents of tumor necrosis factor-α (TNF-α) and induced nitric oxide synthase (iNOS) in the broncho alveolar lavage fluid (BALF) were assayed by ELISA. The expression of M1 polarization markers (CD86，iNOS), myeloid differentiation factor 88(MyD88) and nuclear factor-κB p65(NF-κB p65) in AM were detected by Western blot and quantitative real time-PCR, separately. The distribution and expression of CD86 in the lung were detected by immunohistochemistry. RESULTS: Following modeling, the levels of FVC, FEV0.1, FEV0.3, ratios of FVE0.1/FVC and FEV0.3/FVC were significantly decreased (P<0.01), while the contents of TNF-α and iNOS in the BALF, expression of CD86, iNOS, MyD88 and NF-κB p65 mRNAs and proteins in the AM, and CD86 immunoactivity in the lung were significantly increased in the model group relevant to the normal group (P<0.01). After the intervention, the decrease of the lung function and increase of the above-mentioned genes and proteins were all reversed in the COPD＋EA group (P<0.05, P<0.01). CONCLUSION: EA at ST36 and BL13 can reduce pulmonary inflammation in COPD rats, which may be related to its function in inhibiting M1 polarization of AM via down-regulating MyD88/NF-κB p65 signaling pathway.

Andrographolide attenuates oxidative stress injury in cigarette smoke extract exposed macrophages through inhibiting SIRT1/ERK signaling.

Andrographolide (AG), an ingredient extracted from traditional Chinese herbal medicine Andrographis paniculata, has been demonstrated to have potent anti-inflammatory and anti-oxidative stress properties. The purpose of this study was to investigate whether and how AG attenuated CSE-induced mitochondrial dysfunction, inflammation and oxidative stress in RAW 264.7 cells (a mouse macrophages line). The results showed that AG significantly reduced CSE-induced upregulation of pro-inflammatory cytokines (i.e., TNF-α and IL-1ß) in the RAW 264.7 cells. AG inhibited CSE-induced production of reactive oxygen species (ROS) and prevented the reduction of superoxide dismutase (SOD) and glutathione/oxidized glutathione (GSH/GSSG) ratio, indicating the anti-oxidative stress effects of AG in macrophages. AG also improved mitochondrial function and mitochondrial membrane potential. In addition, AG inhibited CSE-induced increase of heme oxygenase (HO)-1, matrix metalloproteinase (MMP)-9 and MMP-12. Moreover, AG increased SIRT1 transcription and expression, suggesting AG inhibits mitochondrial dysfunction, inflammation and oxidative stress via a SIRT1 dependent signaling. We also demonstrated that AG inhibited CSE-induced ERK phosphorylation, and treatment with PD980589, a ERK inhibitor, reversed CSE-induced inflammation and oxidative stress. These results indicated that AG may prevent COPD via the inhibition of SIRT1/ERK signaling pathway, and subsequently inhibition of mitochondrial dysfunction, inflammation, and oxidative stress in macrophages.

[Macrophages polarization and their role in chronic obstructive pulmonary disease].

Macrophages are highly plastic and can be polarized into classical activated macrophages (M1) and alternative activated macrophages (M2) under the induction of inflammatory factors and regulation of a variety of information molecules. Chronic pulmonary inflammation and pulmonary parenchyma injury are the main pathological manifestations of chronic obstructive pulmonary disease (COPD). M1 promotes pulmonary inflammation, whereas M2 inhibits inflammatory response, participates in lung tissue injury and repair, and swallows and removes pathogenic microorganisms and apoptotic cells. Target intervention in the polarization direction of macrophages may be a new strategy for COPD treatment.

[Effect of moxibustion at "Guanyuan" (CV 4) and "Sanyinjiao" (SP 6) on bone morphology, metabolism and ERα of bone marrow mesenchymal stem cells in the ovariectomized rats].

OBJECTIVE: To observe the effects of moxibustion at "Guanyuan" (CV 4) and "Sanyinjiao" (SP 6) on bone morphology, metabolism and ERα of bone marrow mesenchymal stem cells (MSCs) in the ovariectomized rats and explore the underlying mechanism of moxibustion at Guanyuan (CV 4) and Sanyinjiao (SP 6) on the regulation of bone metabolism. METHODS: A total of 60 SD rats were randomized into a normal group (20 rats) and an ovariectomy group (40 rats). In the normal group, no any treatment was provided. In the ovariectomy group, the classical ovariectomy was adopted to set up the osteoporosis model. In the 13th week after ovariectomy, 10 rats were collected from each of the normal group and the ovariectomy group for model identification. After model identification, the ovariectomy group was subdivided into a model group, an estradiol group and a moxibustion group, 10 rats in each one. In the normal group and the model group, the gavage was provided with 2 mL 0.9% sodium chloride solution, once a day. In the estradiol group, the gavage was provided with 17ß-estradiol 100 µg/(kg•d), once a day. In the moxibustion group, moxibustion was applied at "Guanyuan" (CV 4) and "Sanyinjiao" (SP 6), 15 min at each acupoint, once a day. The 6 treatments were as one course, with 1 day of interval, 12 courses were required. After 12-week intervention, the dual-energy X-ray was adopted to determine the bone mineral density (BMD) and bone mineral content of the whole body in living condition. After sacrificed, HE staining was adopted to observe femur structure, the enzyme-linked immunosorbent assay was to determine the absorbance of estradiol (E2) and alkaline phosphatase (ALP) in serum and the real-time quantitative PCR method was to determine the mRNA expressions of ERα of MSCs in tibia and femur. RESULTS: Compared with the model group, BMD was increased obviously in the pelvis and spine in the moxibustion group and the estradiol group (P<0.05, P<0.01). Compared with the model group, the bone mineral content was higher in the rib, pelvis and spine in the moxibustion group and the estradiol group, there was no statistically significant difference (all P>0.05). Compared with the model group, the bone trabeculas were stronger and well connected in the moxibustion group, indicated by HE staining. Compared with the model group, the concentrations of E2 in serum were increased obviously in the estradiol group and the moxibusiton group (both P<0.01), and ALP concentrations reduced obviously in the estradiol group and the moxibustion group (both P<0.001), the mRNA expressions of ERα in MSCs increased in the estradiol group and the moxibustion group (both P<0.001). CONCLUSION: Moxibustion at "Guanyuan" (CV 4) and "Sanyinjiao" (SP 6) effectively increases BMD and bone strength in the ovariectomized rats and the mechanism may be related to the improvement of serum E2 concentration, the decrease of serum ALP concentration and the up-regulation of mRNA expression of ERα in MSCs.

[Electroacupuncture Improves Pulmonary Function of Rats with Chronic Obstructive Pulmonary Disease by Down-regulating Inflammatory Reaction and Expression of Macrophage Migration Inhibitory Factor/CD 74-CD 44/p 38 MAPK Signaling in Lung Tissues].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Zusanli" (ST 36) and "Feishu" (BL 13) on pulmonary function, inflammatory reaction and expression of macrophage migration inhibitory factor (MIF) and its receptor complex CD 74-CD 44, etc. in rats with chronic obstructive pulmonary disease (COPD), so as to explore its mechanism underlying improvement of COPD. METHODS: Thirty male SD rats were randomly divided into normal, model and EA groups (n＝10 in each group). The COPD model was established by intratracheal infusion of Lipopolysaccharide (LPS, 1 mg/mL) and forced smoke-inhaling. EA was applied to bilateral ST 36 and BL 13 for 30 min, once daily for 7 days. The rat's lung function (forced vital capacity [FVC], forced expiratory capacity ratio ([FEV 0.1/FVC] and [FEV 0.3/FVC]) was detected under anesthesia. Pathological changes of the lung tissue were detected by H．E. staining, and the contents of MIF, tumor necrosis factor-α (TNF-α), interleukin-1 ß (IL-1 ß) and IL-8 in serum, bronchoalveolar lavage fluid (BALF) and lung tissue were assayed by ELISA. The immunoactivity of CD 74 and CD 44 was detected by immunohistochemistry, and the expression levels of MIF, CD 74, CD 44 and p 38 MAPK mRNAs and proteins were examined by quantitative RT-PCR and Western blot, respectively. RESULTS: Compared with the normal group, the FVC, FEV 0.1, FEV 0.3, FEV 0.1/FVC and FEV 0.3/FVC levels were significantly decreased in the model group (P<0.01). After EA treatment, the FVC, FEV 0.1, FEV 0.3, FEV 0.1/FVC and FEV 0.3/FVC were significantly increased (P<0.01, P<0.05), suggesting an improvement of the pulmonary function after EA. H．E. staining showed that the severity of modeling induced alveolar expansion and inflammatory cell infiltration in the lung tissue was relatively milder in the EA group relevant to the model group. The contents of MIF, TNF-α， IL-1 ß and IL-8 in the serum, BALF and lung tissues were significantly higher in the model group than in the normal group (P<0.01), and significantly down-regulated in the EA group relevant to the model group (P<0.01). The expression levels of MIF, CD 74, CD 44 and p 38 MAPK mRNAs and proteins and the immunoactivity levels of CD 74, CD 44 in the lung tissue were obviously higher in the model group than those in the normal group (P<0.01), and considerably lower in the EA group than those in the model group (P<0.01). There was a positive correlation between p 38 MAPK and MIF in mRNA and protein expression levels (P<0.01). CONCLUSION: EA intervention can improve the pulmonary function in COPD rats, which may be related to its effects in inhibiting inflammatory reaction, and MIF/CD 74-CD 44/p 38 MAPK signaling pathway.

Electro-acupuncture regulates the cholinergic anti-inflammatory pathway in a rat model of chronic obstructive pulmonary disease.

OBJECTIVE: Acupuncture has a definite therapeutic effect on chronic obstructive pulmonary disease (COPD), and the cholinergic anti-inflammatory pathway (CAP) has been shown to be involved in regulation of inflammation. In this study, we investigated whether electro-acupuncture (EA) affects the CAP in COPD. METHODS: Sprague-Dawley rats were induced into COPD through exposure to cigarette smoke combined with lipopolysaccharide. EA treatment was applied at Zusanli (ST36) and Feishu (BL13) points for 30 min/d for 7 d. Seventy-two rats were randomly divided into six study groups, including normal, normal + EA, normal + α-bungarotoxin (α-BGT) (the antagonist of the nicotinic acetylcholine receptor α7 subunit (α7nAChR)) + EA, COPD, COPD + EA, and COPD + α-BGT + EA. Lung function, pathology and vagus nerve discharge were tested. The levels of acetylcholine (ACh), acetylcholinesterase (AChE), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in bronchoalveolar lavage fluid (BALF) and lung tissue were measured by enzyme-linked immunosorbent assay. The mRNA and protein expression and immunoreactivity of α7nAChR and its postreceptor inflammation signal pathway, including janus kinase 2 (JAK2), signal transducers and activators of transcription 3 (STAT3), nuclear factor-κB (NF-κB), were observed by quantitative reverse transcription-polymerase chain reaction, Western blot and immunohistochemistry. RESULTS: Compared with normal rats, there were a significant decline in lung function and discharge of the vagus nerve (P < 0.01), a marked sign of lung inflammation and an increase of ACh, AChE, IL-6 and TNF-α level in BALF or lung tissue (P < 0.05, P < 0.01) and higher expression of α7nAChR, JAK2, STAT3 and NF-κB (P < 0.05, P < 0.01) in the COPD rats. In rats receiving EA, the lung function and vagal discharge were enhanced (P < 0.01), lung inflammation was improved and the levels of ACh, AChE, IL-6 and TNF-α were decreased (P < 0.01). Further, the expression of α7nAChR, JAK2, STAT3 and NF-κB was downregulated (P < 0.05, P < 0.01). However, the above effects of EA were blocked in rats injected with α-BGT (P < 0.01). CONCLUSION: EA treatment can reduce the lung inflammatory response and improve lung function in COPD, which may be related to its involvement in the regulation of CAP.

Electroacupuncture reduces hypothalamic and medullary expression of orexins and their receptors in a rat model of chronic obstructive pulmonary disease.

OBJECTIVES: Decreased lung function in chronic obstructive pulmonary disease (COPD) is correlated with abnormal excitability of the respiratory centre where orexin neuropeptides from the hypothalamus are responsible for regulating respiration. We hypothesised that improvements in pulmonary function with electroacupuncture (EA) may be related to orexins in a rat model of COPD. METHODS: The COPD model was established by cigarette smoke exposure and lipopolysaccharide injection. Modelled rats received EA at BL13 and ST36 for two weeks, after which lung function was tested. Orexin levels in the hypothalamus and medulla were detected by ELISA, while mRNA/protein expression and localisation of orexins and their receptors were investigated using real time PCR, Western blotting and immunohistochemistry, respectively. RESULTS: The decrease in lung function observed in COPD rats was improved after EA treatment. Orexin levels in the hypothalamus and medulla were significantly higher in COPD rats than in normal rats, but were significantly reduced in the EA-treated group. There was a negative correlation between orexin content and lung function. In the hypothalamus, mRNA and protein expression and immunoreactivity of orexins were significantly higher in the COPD group than in the normal group, but a significant decrease was observed after EA. In the medulla, the expression and immunoreactivity of orexin receptors were significantly higher in the COPD group than in the normal group, but a significant decrease was observed after EA. CONCLUSIONS: The positive effect of EA on pulmonary function in COPD rats may be related to downregulation of orexins and their receptors in the medulla.

Electroacupuncture at Feishu (BL13) and Zusanli (ST36) down-regulates the expression of orexins and their receptors in rats with chronic obstructive pulmonary disease.

OBJECTIVE: Inflammation and lung function decline are the main pathophysiological features of chronic obstructive pulmonary disease (COPD). Acupuncture can improve lung function in patients with COPD, but the underlying mechanisms are not well understood. Orexins (OXs), which are found in peripheral plasma, are neuropeptides that regulate respiration and their levels are related to COPD. Therefore, we hypothesized that acupuncture might alter OXs, reduce lung inflammation and improve lung function in COPD. METHODS: COPD was induced in rats by exposure to cigarette smoke for 8 weeks and injecting with lipopolysaccharide twice. Electroacupuncture (EA) was performed at Feishu (BL13) and Zusanli (ST36) for 30 min/d for 2 weeks. Rat lung function and morphology were assessed after EA. The levels of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in bronchoalveolar lavage fluid (BALF) and orexin A and B levels in the lung tissue were detected by enzyme-linked immunosorbent assay. OX receptor mRNA levels and immunopositive cells were assessed with real-time polymerase chain reaction and immunohistochemical methods, respectively. The relationships among lung function, cell factors, and OX levels were analyzed by Pearson correlation analyses. RESULTS: Compared with the control group, lung function was significantly decreased in the rats with COPD (P<0.05). There were increases in TNF-α and IL-1ß levels in BALF (P<0.05 and P<0.01, respectively), orexin A level in lung tissue (P<0.01; but not orexin B) and mRNA expressions of OX (OXR1) and OX 2 (OXR2) in lung tissue (P<0.05 and P<0.01, respectively); the integrative optical densities (IODs) of both receptors were greater in the COPD group (P<0.05). For rats with COPD subjected to EA, lung function was improved (P<0.05). There were notable decreases in TNF-α and IL-1ß levels (P<0.05 and <0.01, respectively) in BALF. Orexin A, but not orexinB, levels in lung tissue also decreased (P<0.01), as did mRNA expression of OX1R and OX2R in lung tissue (P<0.05 and P<0.01, respectively). Receptor IODs were also reduced after EA treatment (P<0.05). Furthermore, orexin A levels and ratio of forced expiratory volume in 0.3 s to forced vital capacity were strongly negatively correlated (P<0.01), and orexin A was positively correlated with TNF-α and IL-1ß (P<0.001 and P<0.05, respectively). CONCLUSION: EA at Zusanli and Feishu improved lung function of rats with COPD and had an anti-inflammatory effect, which may be related to down-regulation of OXA and its receptors.

[Protective effect of electroacupuncture intervention on neurovascular unit in rats with focal cerebral ischemia-reperfusion injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) intervention on behavior changes, expression of cerebral vascular endothelial growth factor (VEGF), nerve growth associated protein-43 (GAP-43), synaptophysin (SYN), myelin basic protein (MBP), neurite outgrowth inhibitor-A (Nogo-A) in cerebral focal ischemia-reperfusion injury (CI/RI) rats, so as to study its mechanism underlying improvement of ischemic cerebral vascular disease. METHODS: Sixty male SD rats were randomly divided into sham-operation group, model group and electroacupuncture (EA) group. CI/RI model was established by occlusion of the middle cerebral artery (MCAO) and reperfusion. EA was applied to bilateral "Neiguan" (PC 6), "Sanyinjiao" (SP 6), "Shuigou" (GV 26) and "Baihui" (GV 20) for 30 min, once a day for 14 days. The neurologic deficits were evaluated by Longa 5-grade standard (the higher the score, the severer the neurologic deficit). The immunoactivity of cerebral VEGF, GAP-43, SYN, MBP (important in the process of myelination of nerves in the nervous system) and Nogo-A (inhibiting axonal regeneration) in the focal ischemic cerebral tissue was detected by immunohistochemistry. RESULTS: The Longa's score of the model group was significantly increased after MCAO in comparison with the sham-operation group (P < 0.01). In comparison with the model group, Longa's score of the EA group was evidently lower on day 14 after CI/RI (P < 0.05), suggesting an improvement of the neurological deficits after EA intervention. In comparison with the sham-operation group, the immunoactivity of cerebral VEGF, GAP-43 and Nogo-A was significantly upregulated on day 7 and 14 in the model group (P < 0.01), while that of cerebral SYN was remarkably down-regulated in the model group on day 7 and 14 after CI/RI (P < 0.05). Compared with the model group, cerebral VEGF, GAP-43, SYN and MBP expression levels were considerably upregulated on day 7 and 14 following CI/RI in the EA group (P < 0.01, P < 0.05), while that of cerebral Nogo-A was significantly decreased at the two time-points in the EA group (P < 0.01). CONCLUSION: EA intervention can effectively improve neurological function in cerebral infarction rats, which is closely related to its effects in upregulating cerebral VEGF, GAP-43, SYN and MBP expression, and down-regulating Nogo-A protein, suggesting a protective effect on neurovascular unit.

Effects of electroacupuncture at Zusanli (ST36) on inflammatory cytokines in a rat model of smoke-induced chronic obstructive pulmonary disease.

OBJECTIVE: Improvement in lung function was reported after acupuncture treatment of chronic obstructive pulmonary disease (COPD), but little is known about the underlying mechanisms. Because an immune response imbalance could be seen in COPD, we hypothesize that electroacupuncture (EA) may play a role in regulating inflammatory cytokines and contribute to lung protection in a rat model of smoke-induced COPD. METHODS: A COPD model using male Sprague-Dawley rats exposed to cigarette smoke was established. The rats were randomly divided into four groups (control, sham, COPD, and COPD plus EA), and COPD model was evaluated by measuring pulmonary pathological changes and lung function. EA was applied to the acupuncture point Zusanli (ST36) for 30 min/d for 14 d in sham and COPD rats. Bronchoalveolar lavage fluid (BALF) was used to measure levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and malonaldehyde (MDA). RESULTS: Compared with the control rats, COPD rats had significant changes in lung resistance (RL) and lung compliance (CL) (both P<0.01), bronchi and bronchiole airway obstruction (P<0.01), and levels of MDA, TNF-α, and IL-1ß (P<0.01). There were no significant differences between the control and the sham groups. Compared with the COPD rats, the COPD plus EA rats had decreased RL and increased CL (both P<0.05), and reduced bronchi and bronchiole airway obstruction (P<0.05, P<0.01, respectively), while levels of TNF-α, IL-1ß, and MDA in BALF were lowered (P<0.05 and P<0.01, respectively). However, TNF-α and IL-1ß levels of the EA group rats remained higher than those of the control group (P<0.05). CONCLUSION: EA at ST36 can reduce lung injury in a COPD rat model, and beneficial effects may be related to down-regulation of inflammatory cytokines. The anti-inflammatory and antioxidant effects may prolong the clinical benefit of EA.

Orexin-A and respiration in a rat model of smoke-induced chronic obstructive pulmonary disease.

1. Orexins are neuropeptides synthesized in the hypothalamus that regulate many physiological functions, including energy homeostasis, stress responses, sleep/wake states etc. It is now emerging that orexins may also regulate breathing, but little is known as to how they do this, particularly in chronic obstructive pulmonary disease (COPD). In the present study, we used a rat model of cigarette smoke-induced COPD to investigate orexin-A expression in the hypothalamus and medulla and its effect on respiration. 2. Sprague-Dawley rats were exposed to cigarette smoke (1 h twice daily) for 12 weeks. Lung function and pathological changes associated with inflammation and emphysema were determined to confirm the validity of the COPD model. 3. Hypothalamic and medullary orexin-A levels, as determined by radioimmunoassay, were higher in smoke-exposed than control rats. Furthermore, the expression of prepro-orexin (PPO) mRNA in the hypothalamus and orexin OX(1) receptor mRNA in the medulla, as determined by real-time quantitative polymerase chain reaction, was higher in smoke-exposed than control rats. 4. The number of orexin-A-positive neurons in the hypothalamus and OX(1) and OX(2) receptor-positive neurons in the ventrolateral medulla was higher in smoke-exposed than control rats. 5. Microinjection of orexin-A (1 µmol/L, 0.1 µL) into the pre-Bötzinger complex enhanced phrenic nerve discharge to a greater extent in smoke-exposed compared with control rats (61% vs 36%, respectively). 6. The findings of the present study demonstrate that the increased respiratory activity in smoke-exposed rats is due to an increase in orexin-A as well as upregulation of orexin receptors in the ventrolateral medulla.