OBJECTIVES: To evaluate the protective effect of Bufei Yishen Formula (BYF) against cigarette smoke extract (CSE)-induced injuries in human bronchial epithelial BEAS-2B cells and explore the underlying mechanism. METHODS: BEAS-2B cells exposed to CSE were treated with normal rat serum, BYF-medicated rat serum at low or high doses, pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor), PDTC combined with high-dose BYF-medicated serum, or S-carbomethyloysteine (S-CMC, as the positive control). CCK-8 assay was used to determine the optimal concentration and treatment time of CSE, BYF-medicated serum and S-CMC. The treated cells were examined for inflammatory factor levels in the supernatant and cellular expressions of MUC5AC and MUC5B using ELISA, cell ultrastructural changes with transmission electron microscopy, and cell apoptosis rate using flow cytometry. The expression levels of TLR4/NF‑κB pathway-associated mRNAs and proteins were determined by qRT-PCR and Western blotting. RESULTS: CSE exposure significantly increased secretions of IL-1β, IL-6 and TNF-α, mRNA and protein expressions of MUC5AC and MUC5B, and early and total apoptosis rates in BEAS-2B cells, where the presence of apoptotic bodies was detected. CSE also significantly enhanced the mRNA and protein expressions of TLR4, I-κB, and NF-κB and reduced mRNA and protein expressions of AQP5. Treatments of the CSE-exposed cells with BYF-medicated serum, PDTC and S-CMC all significantly lowered inflammatory factor levels, MUC5AC and MUC5B expressions, and early and total cell apoptosis rates, and partly reversed the changes in cellular ultrastructure and mRNA and protein expressions of the TLR4/NF-κB pathway, and the effects were the most conspicuous following the combined treatment with high-dose BYF-medicated serum and PDTC. CONCLUSIONS BYF can inhibit cell apoptosis, inflammation and mucus hypersecretion in CSE-induced BEAS-2B cells by inhibiting the TLR4/NF-κB signaling pathway.
Humans ; Epithelial Cells/cytology* ; Drugs, Chinese Herbal/pharmacology* ; NF-kappa B/metabolism* ; Bronchi/cytology* ; Smoke/adverse effects* ; Apoptosis/drug effects* ; Mucin 5AC/metabolism* ; Cell Line ; Toll-Like Receptor 4/metabolism* ; Mucin-5B/metabolism* ; Signal Transduction/drug effects* ; Nicotiana ; Rats ; Thiocarbamates/pharmacology* ; Animals

AIM:This study aimed to explore the mechanism by which Bufei-Yishen formula(BYF)mitigates mitochondrial damage in rats with chronic obstructive pulmonary disease(COPD)by regulating the AMPK/PGC-1α signal-ing pathway.METHODS:Forty rats were randomly divided into four groups,each containing ten rats each:control group,COPD group,BYF group,and N-acetylcysteine(NAC)group.The COPD model was established through chronic cigarette smoke exposure combined with periodic bacterial inoculations over an eight-week induction phase.During the subsequent eight-week treatment period(i.e.,weeks 9～16),rats in the control and COPD groups received an isovolumet-ric saline solution via oral gavage,at a standardized daily dose of 2 mL per animal.Moreover,rats in the BYF and NAC groups were given Bufei Yishen formula(11.61 g·kg-1·d-1)or N-acetylcysteine(54 mg·kg-1·d-1)by gavage,once per day.At week 16,samples were collected and the general condition of the rats was observed.Body weight was recorded weekly.We also obtained data characterizing rat lung function,lung pathology,ATP content,and mitochondrial ultra-structure,as well as the levels of interleukin-6(IL-6),tumor necrosis factor-alpha(TNF-α),interleukin-1β(IL-1β),se-rum transforming growth factor-beta 1(TGF-β1)and the enzymatic activities of mitochondrial electron transport chain complexes I(NADH dehydrogenase)and III(cytochrome c reductase).Finally,we quantified the mRNA and protein lev-els of AMPK and PGC-1α in lung tissue.RESULTS:Compared to the control group,the COPD group exhibited yellow-ish hair color,reduced gloss,slower weight gain,and a disordered respiratory rhythm.We also observed significant de-creases(P<0.01)in pulmonary function tidal volume(TV),minute ventilation(MV),peak expiratory flow(PEF),expi-ratory flow at 50%of tidal volume(EF50),forced vital capacity(FVC),forced expiratory volume in 0.1 s(FEV0.1),and FEV0.1/FVC.Histopathological analysis showed alveolar cavity enlargement,bullous changes in lung morphology,smooth muscle hypertrophy in the tracheal wall,ciliary destroyed,mucosal shrinking and thickening,and a large number of in-flammatory cells gathered around the tube.Moreover,the mean linear intercept(MLI)and bronchial wall thickness(BWt)had both significantly increased(P<0.01).Electron microscopic analysis of the lungs revealed a reduction in the number of mitochondria in alveolar epithelial cells,a swollen and deformed lung morphology overall.We observed that the mitochondrial cristae were broken,dissolved or vacuolated,accompanied by a significant reduction in the number of lamel-lar bodies and lung volume,along with a disordered internal lipid layer structure.Furthermore,some lung samples were vacuolated or had content leakage.Further quantitative analyses showed statistically significant increases(P<0.01)in the levels of serum pro-inflammatory mediators,including IL-6,TNF-α,IL-1β,and TGF-β1.At the same time we observed substantial reductions in the enzymatic activities of mitochondrial electron transport chain complexes I and III(P<0.01).Moreover,we found that metabolic impairment correlated with significantly attenuated ATP production(P<0.01)in exper-imental subjects.Moreover,the expression levels of AMPK and PGC-1α mRNA and proteins in lung tissue were signifi-cantly decreased(P<0.01).Moreover,compared to the COPD group,the BYF group showed significant improvements in several of the above indicators,albeit to different degrees(P<0.01 or P<0.05).Moreover,BYF was more effective than NAC in improving minute ventilation and up-regulating PGC-1α expression(P<0.05).CONCLUSION:Bufei-Yishen formula may ameliorate mitochondrial damage in rats with chronic obstructive pulmonary disease by regulating the AMPK/PGC-1α signaling pathway.

AIM:This study aimed to explore the mechanism by which Bufei-Yishen formula(BYF)mitigates mitochondrial damage in rats with chronic obstructive pulmonary disease(COPD)by regulating the AMPK/PGC-1α signal-ing pathway.METHODS:Forty rats were randomly divided into four groups,each containing ten rats each:control group,COPD group,BYF group,and N-acetylcysteine(NAC)group.The COPD model was established through chronic cigarette smoke exposure combined with periodic bacterial inoculations over an eight-week induction phase.During the subsequent eight-week treatment period(i.e.,weeks 9～16),rats in the control and COPD groups received an isovolumet-ric saline solution via oral gavage,at a standardized daily dose of 2 mL per animal.Moreover,rats in the BYF and NAC groups were given Bufei Yishen formula(11.61 g·kg-1·d-1)or N-acetylcysteine(54 mg·kg-1·d-1)by gavage,once per day.At week 16,samples were collected and the general condition of the rats was observed.Body weight was recorded weekly.We also obtained data characterizing rat lung function,lung pathology,ATP content,and mitochondrial ultra-structure,as well as the levels of interleukin-6(IL-6),tumor necrosis factor-alpha(TNF-α),interleukin-1β(IL-1β),se-rum transforming growth factor-beta 1(TGF-β1)and the enzymatic activities of mitochondrial electron transport chain complexes I(NADH dehydrogenase)and III(cytochrome c reductase).Finally,we quantified the mRNA and protein lev-els of AMPK and PGC-1α in lung tissue.RESULTS:Compared to the control group,the COPD group exhibited yellow-ish hair color,reduced gloss,slower weight gain,and a disordered respiratory rhythm.We also observed significant de-creases(P<0.01)in pulmonary function tidal volume(TV),minute ventilation(MV),peak expiratory flow(PEF),expi-ratory flow at 50%of tidal volume(EF50),forced vital capacity(FVC),forced expiratory volume in 0.1 s(FEV0.1),and FEV0.1/FVC.Histopathological analysis showed alveolar cavity enlargement,bullous changes in lung morphology,smooth muscle hypertrophy in the tracheal wall,ciliary destroyed,mucosal shrinking and thickening,and a large number of in-flammatory cells gathered around the tube.Moreover,the mean linear intercept(MLI)and bronchial wall thickness(BWt)had both significantly increased(P<0.01).Electron microscopic analysis of the lungs revealed a reduction in the number of mitochondria in alveolar epithelial cells,a swollen and deformed lung morphology overall.We observed that the mitochondrial cristae were broken,dissolved or vacuolated,accompanied by a significant reduction in the number of lamel-lar bodies and lung volume,along with a disordered internal lipid layer structure.Furthermore,some lung samples were vacuolated or had content leakage.Further quantitative analyses showed statistically significant increases(P<0.01)in the levels of serum pro-inflammatory mediators,including IL-6,TNF-α,IL-1β,and TGF-β1.At the same time we observed substantial reductions in the enzymatic activities of mitochondrial electron transport chain complexes I and III(P<0.01).Moreover,we found that metabolic impairment correlated with significantly attenuated ATP production(P<0.01)in exper-imental subjects.Moreover,the expression levels of AMPK and PGC-1α mRNA and proteins in lung tissue were signifi-cantly decreased(P<0.01).Moreover,compared to the COPD group,the BYF group showed significant improvements in several of the above indicators,albeit to different degrees(P<0.01 or P<0.05).Moreover,BYF was more effective than NAC in improving minute ventilation and up-regulating PGC-1α expression(P<0.05).CONCLUSION:Bufei-Yishen formula may ameliorate mitochondrial damage in rats with chronic obstructive pulmonary disease by regulating the AMPK/PGC-1α signaling pathway.

AIM:To investigate the effects of the three Tiao-Bu Fei-Shen therapies on renal injury in rats with chronic obstructive pulmonary disease(COPD),and to explore the mechanisms.METHODS:SPF-grade SD rats were randomly divided into control,COPD,pyrrolidinedithiocarbamate ammonium(PDTC),Bufei-Jianpi formula(BJF),Bu-fei-Yishen formula(BYF),Yiqi-Zishen formula(YZF),BJF combined with PDTC(BJF+PDTC),BYF combined PDTC(BYF+PDTC),and YZF combined PDTC(YZF+PDTC)groups.Cigarette smoke exposure combined with bacterial infec-tion were used to develop a stable-phase rat model of COPD,and kidney-injured rats were screened for subsequent treat-ment.Pulmonary and renal functions,pathological changes in lung and kidney tissues,24 h urine volume,urine biochem-ical indexes,aquaporin(AQP)levels,ratio of inflammatory cells in the bronchoalveolar lavage fluid(BALF),serum in-flammatory factor levels,and mRNA and protein expressions of IκB kinase(IKK)and nuclear factor-κB(NF-κB)were observed at the end of the 16th week.RESULTS:Compared with control group,the rats in COPD group showed reduced lung function indexes of forced expiratory vital capacity(FVC),forced expiratory volume in 0.1 second(FEV0.1)and FEV0.1/FVC(P<0.05).The lung histopathology exhibited alveolar wall fracture and fusion and airway inflammatory cell infiltration.The renal function indexes serum creatinine,blood urea nitrogen and serum cystatin C(Cys-C)were signifi-cantly increased(P<0.01).The renal histopathology showed swollen and disorderly arranged tubular epithelial cells.The 24 h urine volume decreased(P<0.01).The urinary biochemical indexes 24 h urinary total protein,urinary kidney injury molecule-1(KIM-1),urinary Cys-C,and urinary N-acetyl-β-D-glucosaminidase significantly increased(P<0.01).The protein levels of AQP1～4 were significantly increased(P<0.01).The ratio of neutrophils and lymphocytes in the BALF were increased(P<0.01).The ratio of monocytes was decreased(P<0.01).The serum levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6,IL-13,IL-1β,and transforming growth factor-β1(TGF-β1)were significantly increased(P<0.01).And the mRNA and protein expression levels of IKK,NF-κB were significantly elevated in the renal tissue(P<0.05).Compared with COPD group,the above symptoms and indexes of the rats were improved to different degrees after the intervention in treatment groups,among which BJF,BYF and YZF demonstrated similar effects to PDTC,and BJF+PDTC,BYF+PDTC and YZF+PDTC exhibited better improvements than the corresponding monotherapy.CONCLU-SION:The three Tiao-Bu Fei-Shen therapies can reduce the airway and systemic inflammatory response,improve renal function and attenuate renal injury in COPD rats.The mechanism may be related to the inhibition of NF-κB signaling path-way.

Objective To investigate the roles of three Tiao-Bu Fei-Shen Traditional Chinese Medicine(TCM)therapies in improving airway mucus hypersecretion in rats with stable chronic obstructive pulmonary disease(COPD).Methods Ninety rats were divided randomly into nine groups:control(Control)group,model(COPD)group,Bu-Fei Jian-Pi Formula(BJF)group,Bu-Fei Yi-Shen Formula(BYF)group,Yi-Qi Zi-Shen Formula(YZF)group,ERK1/2 inhibitor(PD98059)group,Bu-Fei Jian-Pi combined with inhibitor(BJF+PD98059)group,Bu-Fei Yi-Shen combined with inhibitor(BYF+PD98059)group,and Yi-Qi Zi-Shen combined with inhibitor(YZF+PD98059)group.A rat model of COPD was established by exposing rats to cigarette smoke followed by repeated bacterial infection from weeks 1～8.From weeks 9～16,rats in the control and COPD groups were given 2 mL normal saline,rats in the BJF,BYF,and YZF groups were given the three Tiao-Bu Fei-Shen formulas by gavage,and rats in the PD98059,BJF+PD98059,BYF+PD98059,and YZF+PD98059 groups were given PD98059 by intraperitoneal injection for 7 days at the 16th week.Lung function tests were conducted after 16 weeks and lung tissue morphology,lung water content,inflammatory cell count in bronchoalveolar lavage fluid,and serum levels of inflammatory factors were also assessed.Goblet cell proportion was determined by Alcian blue-periodic acid-Schiff staining,and Muc5AC and Muc5B expression levels were detected by immunohistochemistry.mRNA expression levels of ERK1,ERK2,ENaC,CFTR,and AQP5 were detected by polymerase chain reaction and protein expression levels of ERK1/2 and P-ERK1/2 in lung tissue were determined by Western Blot.Results TV,MV,FVC,FEV0.1,FEV0.1/FVC were significantly decreased(P＜0.01)in COPD rats compared with those in the control group.Lung pathology revealed alveolar disorder,massive fracture of the alveolar wall,and severe shrinkage/thickening of the airway wall accompanied by extensive infiltration of inflammatory cells.Lung tissue water content was significantly increased in COPD rats(P＜0.01),while the proportion of macrophages in BALF was significantly reduced(P＜0.01)and the proportions of neutrophils and lymphocytes were significantly increased(P＜0.01).Serum levels of TNF-α and IL-1β were significantly increased in COPD rats(P＜0.05,P＜0.01).The percentage of goblet cells and expression levels of Muc5AC and Muc5B in airway epithelial cells were significantly increased(P＜0.01),mRNA expression levels of ERK1,ERK2,and ENaC in lung tissue were significantly elevated(P＜0.01),while mRNA expression levels of CFTR and AQP5 were significantly decreased(P＜0.01)in COPD rats compared with levels in the control group.The expression of P-ERK1/2,ERK1/2 in lung tissue was significantly increased(P＜0.01)Rats in the treatment groups demonstrated improvements in the above indicators(P＜0.05,P＜0.01)compared with the COPD group,the groups receiving the three Tiao-Bu Fei-Shen formulas combined with PD98059 showing superior efficacy compared with the single treatment groups(P＜0.05,P＜0.01).Conclusions The three tested Tiao-Bu Fei-Shen therapies can ameliorate airway mucus hypersecretion in COPD rats by inhibiting the ERK1/2 signaling pathway.

Chronic obstructive pulmonary disease(COPD)is a disease characterized by persistent respiratory symptoms and airflow limitation,and also associated with a variety of comorbidities.As one of the important extrapulmonary comorbidities of COPD,kidney injury has a high incidence and insidious onset,which will develop into chronic kidney disease without timely intervention,greatly increasing the risk of mortality and economic burden of COPD patients.There exist physiological and pathological relations between lung and kidney functions.However,the mechanisms underlying COPD-associated kidney injury remain unclear.This article provides a review of the research progress on the mechanisms of COPD-associated kidney injury,in order to provide reference for the clinical prevention and treatment and basic research.

Objective To investigate the mechanism by which Xuanfei Jiedu Formula(XFJDF)ameliorates pulmonary damage in rats with multidrug-resistant Pseudomonas aeruginosa(MDR-PA)pneumonia,by modulating the activity of the inhibitor of nuclear factor(NF)-κB kinase(IKK)IKK/NF-κB signal transduction cascade.Methods Eighty-four rats were divided randomly into seven groups:control,model,XFJDF-low dose,XFJDF-medium dose,XFJDF-high dose,imipenem(IPM),and pyrrolidinedithiocarbamate ammonium(PDTC)groups(n=12 rats per group).A MDR-PA pneumonia rat model was established by oral tracheal intubation.After successful model construction,rats in the low-,medium-,and high-dose XFJDF groups were given the corresponding dose of drugs by gavage,rats in the IPM group were given an intraperitoneal injection of IPM,and rats in the control and model groups were given the same volume of normal saline by gavage,twice a day for 7 days.PDTC was injected intraperitoneally 1 h before model establishment,12 h and 24 h after model establishment in the NF-κB inhibitor group.The behavior status,body weight changes,spleen and thymus indexes,and lung wet weight/dry weight ratio were observed in the different groups.Histological changes in the lung tissue were assessed by hematoxylin and eosin staining.Terminal deoxynucleotidyl transferase dUTP nick end labeling was used to detect apoptosis of lung tissue cells,and serum levels of interleukin(IL)-1 β,tumor necrosis factor(TNF)-α,transforming growth factor(TGF)-β,and IL-10 were detected by enzyme-linked immunosorbent assay,and glutathione(GSH)and malondialdehyde(MDA)levels,myeloperoxidase(MPO)activity and total antioxidant capacity(T-AOC)were determined by colorimetry and thiobarbituric acid assay.NF-κBp65 in lung tissue was identified by immunohistochemical analysis,IKKβ and NF-κBp65 mRNA were analyzed by reverse transcription quantitative polymerase chain reaction,and expression levels of IKKβ,phosphorylated(p)-IKKβ,NF-κBp65,and p-NF-κBp65 were measured by Western blot.Results Compared with the control group,rats in the model group exhibited decreased appetite,dull fur,sluggish responsiveness,decreased mobility,increased respiratory rate,audible murmurs,and notable weight loss(P＜0.01).The spleen and thymus indices were significantly enhanced(P＜0.01)and the lung wet/dry weight ratio was significantly increased(P＜0.01),concurrent with increased alveolar secretions in lung tissue.Infiltration of abundant inflammatory cells and increased apoptosis in lung tissue were also noted.Serum concentrations of IL-1β,TNF-α,TGF-β,and IL-10 were increased(P＜0.01)and the MDA content and MPO activity were also increased(P＜0.01).Conversely,GSH levels and T-AOC were significantly decreased(P＜0.01).Lung levels of IKKβ and NF-κBp65 mRNA were significantly increased(P＜0.01)and the ratios of p-IKKβ/IKKβ and p-NF-κBp65/NF-κBp65 were also significantly increased(P＜0.01)compared with the control group.XFJDF,IPM and PDTC improved these parameters to varying degrees,compared with the model group(P＜0.05,P＜0.01),with particularly significant effects in the high-dose XFJDF and IPM groups.Conclusions XFJDF may improve lung injury in rats with MDR-PA pneumonia by inhibiting the IKK/NF-κB signaling pathway.

Objective To investigate the mechanism by which Xuanfei Jiedu Formula(XFJDF)ameliorates pulmonary damage in rats with multidrug-resistant Pseudomonas aeruginosa(MDR-PA)pneumonia,by modulating the activity of the inhibitor of nuclear factor(NF)-κB kinase(IKK)IKK/NF-κB signal transduction cascade.Methods Eighty-four rats were divided randomly into seven groups:control,model,XFJDF-low dose,XFJDF-medium dose,XFJDF-high dose,imipenem(IPM),and pyrrolidinedithiocarbamate ammonium(PDTC)groups(n=12 rats per group).A MDR-PA pneumonia rat model was established by oral tracheal intubation.After successful model construction,rats in the low-,medium-,and high-dose XFJDF groups were given the corresponding dose of drugs by gavage,rats in the IPM group were given an intraperitoneal injection of IPM,and rats in the control and model groups were given the same volume of normal saline by gavage,twice a day for 7 days.PDTC was injected intraperitoneally 1 h before model establishment,12 h and 24 h after model establishment in the NF-κB inhibitor group.The behavior status,body weight changes,spleen and thymus indexes,and lung wet weight/dry weight ratio were observed in the different groups.Histological changes in the lung tissue were assessed by hematoxylin and eosin staining.Terminal deoxynucleotidyl transferase dUTP nick end labeling was used to detect apoptosis of lung tissue cells,and serum levels of interleukin(IL)-1 β,tumor necrosis factor(TNF)-α,transforming growth factor(TGF)-β,and IL-10 were detected by enzyme-linked immunosorbent assay,and glutathione(GSH)and malondialdehyde(MDA)levels,myeloperoxidase(MPO)activity and total antioxidant capacity(T-AOC)were determined by colorimetry and thiobarbituric acid assay.NF-κBp65 in lung tissue was identified by immunohistochemical analysis,IKKβ and NF-κBp65 mRNA were analyzed by reverse transcription quantitative polymerase chain reaction,and expression levels of IKKβ,phosphorylated(p)-IKKβ,NF-κBp65,and p-NF-κBp65 were measured by Western blot.Results Compared with the control group,rats in the model group exhibited decreased appetite,dull fur,sluggish responsiveness,decreased mobility,increased respiratory rate,audible murmurs,and notable weight loss(P＜0.01).The spleen and thymus indices were significantly enhanced(P＜0.01)and the lung wet/dry weight ratio was significantly increased(P＜0.01),concurrent with increased alveolar secretions in lung tissue.Infiltration of abundant inflammatory cells and increased apoptosis in lung tissue were also noted.Serum concentrations of IL-1β,TNF-α,TGF-β,and IL-10 were increased(P＜0.01)and the MDA content and MPO activity were also increased(P＜0.01).Conversely,GSH levels and T-AOC were significantly decreased(P＜0.01).Lung levels of IKKβ and NF-κBp65 mRNA were significantly increased(P＜0.01)and the ratios of p-IKKβ/IKKβ and p-NF-κBp65/NF-κBp65 were also significantly increased(P＜0.01)compared with the control group.XFJDF,IPM and PDTC improved these parameters to varying degrees,compared with the model group(P＜0.05,P＜0.01),with particularly significant effects in the high-dose XFJDF and IPM groups.Conclusions XFJDF may improve lung injury in rats with MDR-PA pneumonia by inhibiting the IKK/NF-κB signaling pathway.

The global production and consumption of sugar-sweetened beverages (SSBs) has been on the rise in recent decades. The intake of SSBs has been increasing in China, and it is more prevalent among children and adolescents. As research continues to intensify, more and more studies have shown that, in addition to the increased risks of dental caries and obesity reported by the World Health Organization (WHO), SSBs intake can also increase risks of chronic diseases such as diabetes, cardiovascular disease, gout, and cancer, and early death, adding to the burden of disease. Due to the health risks associated with the overconsumption of SSBs, many countries around the world have taken measures to control the intake. The main measures currently in place are taxation of SSBs, restrictions on marketing and sales of SSBs, front-of-package labeling and reducing availability of SSBs in schools. In China, the main measures currently in place are to control the sales of beverages in schools, with Shenzhen taking the lead in implementing health warning labeling to alcoholic beverages and carbonated beverages, extending the measures to reduce SSBs intake beyond school grounds.

Thyroid carcinoma is a common cervical tumor. Its occurrence is associated with genetic and environmental factors. The incidence rate of thyroid cancer is increasing. However, the pathogenesis and the influencing factors of thyroid carcinoma are not yet fully understood. Many clinical studies and epidemiological investigations have found that such factors as radiation exposure, female hormone metabolism, and obesity have important links with the occurrence of thyroid cancer, but its association with factors such as dietary factors remains controversial. Therefore, it is urgent to explore and validate the relationship between various factors and thyroid cancer through epidemiological studies. In order to provide reference for the prevention of thyroid cancer, this special column “Thyroid carcinoma and associated risk factors” focused on the associations of female reproductive factors and dietary factors (including intake of iodine-rich foods) with thyroid carcinoma (nodules).