Machine Learning Approach in Dosage Individualization of Isoniazid for Tuberculosis.

INTRODUCTION: Isoniazid is a first-line antituberculosis agent with high variability, which would profit from individualized dosing. Concentrations of isoniazid at 2 h (C2h), as an indicator of safety and efficacy, are important for optimizing therapy. OBJECTIVE: The objective of this study was to establish machine learning (ML) models to predict the C2h, that can be used for establishing an individualized dosing regimen in clinical practice. METHODS: Published population pharmacokinetic (PopPK) models for adults were searched based on PubMed and ultimately four reliable models were selected for simulating individual C2h datasets under different conditions (demographics, genotype, ethnicity, etc.). Machine learning models were trained on simulated C2h obtained from the four PopPK models. Five different algorithms were used for ML model building to predict C2h. Real-world data were used for predictive performance evaluations. Virtual trials were used to compare ML-optimized doses with PopPK model-optimized doses. RESULTS: Categorical boosting (CatBoost) exhibited the highest prediction ability. Target C2h can be predicted using the ML model combined with the dosing regimen and three covariates (N-acetyltransferase 2 [NAT2] genotypes, weight and race [Asians and Africans]). Real-world data validation results showed that the ML model can achieve an overall prediction accuracy of 93.4%. Using the final ML model, the mean absolute prediction error value decreased by 45.7% relative to the average of PopPK models. Using the ML-optimized dosing regimen, the probability of target attainment increased by 43.7% relative to the PopPK model-optimized dosing regimens. CONCLUSION: Machine learning models were developed with great predictive performance, which can be used to determine the individualized initial dose of isoniazid in adult patients.

Optimal use of ß-lactams in neonates: machine learning-based clinical decision support system.

BACKGROUND: Accurate prediction of the optimal dose for ß-lactam antibiotics in neonatal sepsis is challenging. We aimed to evaluate whether a reliable clinical decision support system (CDSS) based on machine learning (ML) can assist clinicians in making optimal dose selections. METHODS: Five ß-lactam antibiotics (amoxicillin, ceftazidime, cefotaxime, meropenem and latamoxef), commonly used to treat neonatal sepsis, were selected. The CDSS was constructed by incorporating the drug, patient, dosage, pharmacodynamic, and microbiological factors. The CatBoost ML algorithm was used to build the CDSS. Real-world studies were used to evaluate the CDSS performance. Virtual trials were used to compare the CDSS-optimized doses with guideline-recommended doses. FINDINGS: For a specific drug, by entering the patient characteristics and pharmacodynamic (PD) target (50%/70%/100% fraction of time that the free drug concentration is above the minimal inhibitory concentration [fT > MIC]), the CDSS can determine whether the planned dosing regimen will achieve the PD target and suggest an optimal dose. The prediction accuracy of all five drugs was >80.0% in the real-world validation. Compared with the PopPK model, the overall accuracy, precision, recall, and F1-Score improved by 10.7%, 22.1%, 64.2%, and 43.1%, respectively. Using the CDSS-optimized doses, the average probability of target concentration attainment increased by 58.2% compared to the guideline-recommended doses. INTERPRETATION: An ML-based CDSS was successfully constructed to assist clinicians in selecting optimal ß-lactam antibiotic doses. FUNDING: This work was supported by the National Natural Science Foundation of China; Distinguished Young and Middle-aged Scholar of Shandong University; National Key Research and Development Program of China.

A first-in-human phase 1 study of simnotrelvir, a 3CL-like protease inhibitor for treatment of COVID-19, in healthy adult subjects.

Safe and efficacious antiviral therapeutics are in urgent need for the treatment of coronavirus disease 2019. Simnotrelvir is a selective 3C-like protease inhibitor that can effectively inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated the safety, tolerability, and pharmacokinetics of dose escalations of simnotrelvir alone or with ritonavir (simnotrelvir or simnotrelvir/ritonavir) in healthy subjects, as well as the food effect (ClinicalTrials.gov Identifier: NCT05339646). The overall incidence of adverse events (AEs) was 22.2% (17/72) and 6.3% (1/16) in intervention and placebo groups, respectively. The simnotrelvir apparent clearance was 135-369 L/h with simnotrelvir alone, and decreased significantly to 19.5-29.8 L/h with simnotrelvir/ritonavir. The simnotrelvir exposure increased in an approximately dose-proportional manner between 250 and 750 mg when co-administered with ritonavir. After consecutive twice daily dosing of simnotrelvir/ritonavir, simnotrelvir had a low accumulation index ranging from 1.39 to 1.51. The area under the curve of simnotrelvir increased 44.0 % and 47.3 % respectively, after high fat and normal diet compared with fasted status. In conclusion, simnotrelvir has adequate safety and tolerability. Its pharmacokinetics indicated a trough concentration above the level required for 90 % inhibition of SARS-CoV-2 in vitro at 750 mg/100 mg simnotrelvir/ritonavir twice daily under fasted condition, supporting further development using this dosage as the clinically recommended dose regimen.

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.

Design, synthesis and structure-activity relationship optimization of phenanthridine derivatives as new anti-vitiligo compounds.

Humans have been suffering from vitiligo for a long time. Target vitiligo drugs have yet been approved. Activation of Wnt/ß-catenin signalling has potential in the therapeutic use of vitiligo, so exploring new drugs that specifically directly activate Wnt is worthwhile to obtain new anti-vitiligo agents. In this work, two portions design and synthesis were put into effect. firstly, 17 phenanthridine derivatives with C-4 substitutes were designed and synthesized, which compounds 4, 6, 12, 13 served as H-acceptor with protein showed enhance melanogenesis activity; Secondly, 7 hybrid new scaffolds of compounds were designed and synthesized, scaffold hopping compound 36 that aromatic benzene was replaced pyrazole on ring C showed enhance melanogenesis and tyrosinase activity; The last and most important, a comprehensive optimization and SARs of compound 36 were carried out, compounds 41 and 43 shared phenolic hydroxyl or 3-methyl-pyridine substitutes at C-7 position remarkably improved the capacity of melanogenesis and tyrosinase activity. Compound 43 were identified as new anti-vitiligo agents that specifically activate the Wnt/ß-catenin signalling pathway by targeting Axin. Structure-activity relationship analysis implied that H-acceptor substitutions at the C-4 position and phenolic hydroxyl or pyridine substitutions at the C-7 position would improve the activities of the compounds. These findings reveal a new therapeutic strategy for vitiligo, and compounds 41 and 43 may represent potential compounds for vitiligo treatment.

Novel nucleocapsid protein-targeting phenanthridine inhibitors of SARS-CoV-2.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unprecedented in human history. As a major structural protein, nucleocapsid protein (NPro) is critical to the replication of SARS-CoV-2. In this work, 17 NPro-targeting phenanthridine derivatives were rationally designed and synthesized, based on the crystal structure of NPro. Most of these compounds can interact with SARS-CoV-2 NPro tightly and inhibit the replication of SARS-CoV-2 in vitro. Compounds 12 and 16 exhibited the most potent anti-viral activities with 50% effective concentration values of 3.69 and 2.18 µM, respectively. Furthermore, site-directed mutagenesis of NPro and Surface Plasmon Resonance (SPR) assays revealed that 12 and 16 target N-terminal domain (NTD) of NPro by binding to Tyr109. This work found two potent anti-SARS-CoV-2 bioactive compounds and also indicated that SARS-CoV-2 NPro-NTD can be a target for new anti-virus agents.

Two new cytotoxic cycloartane triterpenoids from Aphanamixis polystachya (Wall.) R. N. Parker.

Two new cycloartane triterpenoids, (24 R)-cycloartane-3ß,24,25,30-tetrol (1) and (24 R)-24,25,30-trihydroxy-9,19-cycloartane-3-one (2), along with three known compounds (3-5) were isolated from leaves and twigs of Aphanamixis polystachya. The new compounds were elucidated based on comprehensive spectroscopic analysis, including 1 D, 2 D NMR and HREIMS. The in vitro cytotoxic activities evaluation of five human cancer cell lines revealed that compound 1 exhibited cytotoxic activity on all of tested human cancer cell lines, while compound 2 only had specific activity on SMMC-7721 cell line.

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.

Daily expression of two circadian clock genes in compound eyes of Helicoverpa armigera: evidence for peripheral tissue circadian timing.

Circadian clock genes in peripheral tissues usually play an important role in regulating the circadian rhythms. Light is the most important environmental signal for synchronizing endogenous rhythms with the daily light-dark cycle, and compound eyes are known as the principal circadian photoreceptor for photic entrainment in most moths. However, there is little evidence for circadian timing in compound eyes. In the current study, we isolated the timeless gene, designated Ha-tim (GenBank accession number: KM233162), from the cotton bollworm Helicoverpa armigera. Ha-tim and period (Ha-per) showed low messenger RNA levels in the compound eyes compared to the other tested adult organs. Ha-tim and Ha-per transcript levels were dependent on an endogenous rhythm that fluctuated over a daily cycle in the compound eyes and heads. The cycles of Ha-tim and Ha-per transcript levels followed similar time courses, and identical expression patterns of the two genes were observed in the compound eyes and heads. Ha-tim and Ha-per were down-regulated in the compound eyes after light exposure, copulation and starvation. These results indicated that Ha-tim and Ha-per transcript levels were regulated by endogenous and exogenous factors. Our study helped to improve our understanding of the circadian clock machinery in compound eyes and other peripheral tissues.

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.

The expression of three opsin genes and phototactic behavior of Spodoptera exigua (Lepidoptera: Noctuidae): Evidence for visual function of opsin in phototaxis.

Phototaxis in nocturnal moths is widely utilized to control pest populations in practical production. However, as an elusive behavior, phototactic behavior is still not well understood. Determination of whether the opsin gene plays a key role in phototaxis is an interesting topic. This study was conducted to analyze expression levels and biological importance of three opsin genes (Se-uv, Se-bl, and Se-lw) and phototactic behavior of Spodoptera exigua. The three opsin genes exhibited higher expression levels during daytime, excluding Se-bl in females, whose expression tended to increase at night. And cycling of opsin gene levels tended to be upregulated at night, although the magnitude of increase in females was lower than that in males exposed to constant darkness. The results of western blotting were consistent with those of qRT-PCR. Furthermore, opsin gene expression was not influenced by light exposure during the scotophase, excluding Se-uv in males, and tended to be downregulated by starvation in females and copulation in both female and male moths. To determine the relationship between opsin gene expression and phototactic behavior, Se-lw was knocked down by RNA interference. Moths with one opsin gene knocked down showed enhanced expression of the other two opsin genes, which may play important roles in compensation in vision. The Se-lw-knockdown moths exhibited reduced phototactic efficiency to green light, suggesting that Se-LW contributes to phototaxis, and increases phototactic efficiency to green light. Our finding provides a sound theoretical basis for further investigation of visual expression pattern and phototactic mechanisms in nocturnal moths.