Objective: To investigate the anti-inflammatory effect of hesperetin (HSP) on lung damage induced by paraquat (PQ) in rats by detecting the levels of inflammatory makers in rat lung tissues. Methods: 140 Wistar male rats were randomly divided into negative control group, HSP control group, HSP control group, paraquat model group, pirfenidone (PDF) positive control group, and 100, 200, 400 mg/kg HSP treatment groups. All groups were exposed to 50mg/kg paraquat by oral gavage except for the negative control group and HSP control group. After 24 hours, the rats in each group were given drug intervention once daily. 10 rats were randomly sacrificed at 7th day and 28th day after exposure to paraquat respectively. 3 rats were randomly selected from them and HE, Masson staining were used to observe the pathological changes in the lungs of each group. Each group randomly selected 6 rats at two time points to detect the levels of TGF-β₁, TNF-α, IL-4, IL-10, IL-1β and IFN-γ in rat lung tissues. Results: Histopathological examination found that the lung injury were reduced in the rats of PDF positive control group and all HSP treatment groups. Compared with the negative control group, the levels of TGF-β1, IL-1β, TNF-α, IL-4, and IL-10 in rat lung tissues were significantly increased (P<0.05, P<0.01) after PQ exposure at two points in time, and there was no significant difference in the level of IFN-γ in lung tissues compared with the negative control group (P>0.05) . The levels of TGF-β1, IL-1β, IL-4, IL-10 and TNF-α in the lung tissues of rats on the 7th day in different dose treatment groups of HSP were reduced compared with those in the PQ model group with varying degrees (P<0.05, P<0.01) . The level of IFN-γ in lung tissues of rats were not significantly different from that of model group (P>0.05) . The levels of TGF-β₁ and TNF-α in lung tissue of rats on the 28th day in PDF positive control group and different dose treatment groups of HSP were reduced compared with those in the PQ model group with varying degrees (P<0.05, P<0.01). The levels of IFN-γ in the rat lung tissues were increased compared with those in the PQ model group (P<0.05). Besides, there were no significant in the levels of IL-1β, IL-4 and IL-10 in lung tissues compared with PQ model group (P>0.05). Conclusion HSP can reduce lung damage induced by PQ in rats by inhibiting the release of inflammatory factors and promoting the secretion of anti-inflammatory factors.

OBJECTIVE: To explore the changes of retinoic acid related orphan receptor-γt( ROR-γt),interleukin( IL)-17 A and forkhead / winged helix protein 3( Foxp3) mRNA expression and promoter methylation in the process of asthma induced by toluene-diisocyanate( TDI). METHODS: Specific pathogens free grade healthy male BALB / c mice were randomly assigned into asthma group and control group with 18 animals in each group. In the asthma group,the mice were sensitized with 0. 30% TDI( mass-volume concentration) dropped on the dorsum of both ears( 20 μL / ear) on day 1 and day 8. On day 15,the mice were challenged with 20 μL 0. 01% TDI( mass-volume concentration) by the trachea. The control group mice were sensitized and challenged by the same procedures with the same amount of solvent( acetone / olive oil). The mice were challenged 24 hours,the pathological changes of trachea and lung tissues were observed. The bronchoalveolar lavage fluid( BALF) from each group was collected,and the inflammatory cells in BALF were counted and classified. IL-4and Interferon-γ( IFN-γ) levels in BALF supernatant were measured by enzyme-linked immunosorbent assay. ROR-γt,IL-17 A and Foxp3 mRNA expression in the lung tissue were measured by real-time fluorescent quantitative polymease chain reaction. The degree of ROR-γt,IL-17 A and Foxp3 promoter methylation in lung issue were detected by Mass Array system.RESULTS: The asthmatic group demonstrated the symptoms of acute asthma,such as breathing deeply and fastly,bowing the back,lifting the forelimbs,et al. But the control group had no such symptoms in mice. Hematoxylin-Eosin staining showed obvious inflammatory lesions in the trachea and lung tissue of asthmatic mice. Compared with the control group,the white blood cell count,the neutrophil and eosinophil percentages in BALF,the IL-4,IFN-γ levels in BALF supernatant in asthma group were all significantly increased( P < 0. 01),meanwhile the lymphocyte and monocyte percentages in BALF were reduced( P < 0. 01); ROR-γt mRNA expression was significantly increased( P < 0. 01),and the degree of promoter methylation from sites 3,4,5,6,8,11 and 12 was significantly reduced( P < 0. 05); IL-17 A mRNA expression was significantly increased( P < 0. 01),and the degree of promoter methylation from sites 6 and 7 was significantly reduced( P < 0. 01); Foxp3 mRNA expression was significantly reduced( P < 0. 01),and the degree of promoter methylation from sites 1 and 10 was significantly increased( P < 0. 01). CONCLUSION: Th17 / Treg cell immune imbalance occurs in asthma induced by TDI. ROR-γt,IL-17 A and Foxp3 gene promoter methylation abnormalities may be involved in Th17 / Treg cell immune imbalance.

Objective: To explore the changes in the autophagy marker microtubule-associated protein 1 light chain 3 (LC3) and yeast autophagy-related gene 6 (Beclin1) in rat lungs exposed to free silica (SiO₂) dust for different periods. Methods: A total of 72 male specific pathogen-free Wistar rats were randomly divided into solvent control group and SiO₂ model group. The SiO₂ model group received one-time non-exposed intratracheal instillation of suspension of SiO₂ particles to establish a model of silicosis. The solvent control group received an equal amount of saline. Six rats each were sacrificed at 1, 7, 14, 21, 28, and 60 days after model establishment. The pathological changes and fibrosis of rat lungs at different time points were evaluated by H&E staining and Masson staining, respectively. Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of transforming growth factor-β (TGF-β) , interleukin-1 (IL-1) , and tumor necrosis factor-α (TNF-α) in lung tissue homogenate. Western blot was used to determine the relative expression levels of LC3 and Beclin1 in the lung tissue. Results: The results of H&E staining showed that the model group had continuous inflammation in the lung tissue from day 1 to day 60, and the inflammatory scores were significantly higher in the model group than in the control group (P<0.05) . The results of Masson staining showed that rats in the model group had a small amount of collagen fibers in the lung tissue on day 14 and a large amount of collagen fibers on day 60. The fibrosis score was significantly higher in the model group than in the control group (P<0.05) . No collagen fibrosis was observed in the lung tissue in the control group. The results of ELISA showed that the model group had significantly higher levels of IL-1, TNF-α, and TGF-β in lung tissue homogenate than the control group at each time point after exposure (P<0.05) . The results of Western blot showed that the model group had decreased expression of Beclin1 protein in the lung tissue on days 7, 14, 21, 28, and 60, which was significantly higher than that of the control group (P<0.05) . The model group also had a decreased ratio of LC3II/LC3I on days 1, 7, 14, 21, 28, and 60, which were significantly higher than that of the control group (P<0.05) . Conclusion In the rat model of silicosis induced by free SiO₂ dust, the expression levels of autophagy-related proteins, LC3 and Beclin1, are correlated with different stages of silicosis. In the early stage of silicosis, the lung tissue has inflammation, substantially increased ratio of LC3II/LC3I and expression of Beclin1, and active autophagy. With the progression of silicosis, the ratio of LC3II/LC3I and expression level of Beclin1 gradually decrease and autophagy becomes weak.

Objective: The aim of this study was to investigate the effect of mitogen-activated protein kinase (MAPK) signaling pathway on apoptosis induced by chloroacetic acid in human normal bronchial epithelial 16HBE cells. Methods: 16HBE cells were exposed to 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 mmol/L chloroacetic acid for 24 h in vitro. The cytotoxicity induced by chloroacetic acid was assessed by CCK-8 and LDH assays. Cell apoptosis was detected by Annexin V-FITC and PI staining. The protein expression levels of phosphorylation of p38, ERK1/2 and JNK were determined by western blotting. 16HBE cells were pretreated with MAPK signaling pathway specific inhibitors including SB203580, U0126 and SP600125 for 1 h, and these cells were subsequently treated with 2.5 mmol/L chloroacetic acid for 24 h. The expressions of p-p38, p-ERK1/2 and p-JNK as well as the changes of cell viability and apoptosis were measured after pretreated with inhibitors for 1 h. Results: The cell viability by CCK-8 and LDH methods gradually reduced in a dose-dependent manner when chloroacetic acid concentrations elevated (P<0.05) , and their correlation coefficients were -0.902 and -0.825, respectively. The detection efficiency of CCK-8 assay significantly increased compared with LDH assay (P<0.05) . The cell apoptosis rates, which were (17.2±4.0) %, (24.6± 4.2) %, (39.3 ± 5.7) % in 1.5, 2.0, 2.5 mmol/L chloroacetic acid-treated groups, were higher than that of the control group[ (5.6 ± 3.0) %] (P<0.05) . There was a time-or dose-dependent change in the protein expressions of p-p38, p-ERK1/2 and p-JNK. Compared with the control, the levels of p-p38 had 2.1 and 2.6-fold increases in 16 and 24 h treated groups (P<0.01) , while the levels of p-ERK1/2 distinctly decreased by 37% and 52% (P<0.01) . In comparison with the control group, the expressions of p-p38 had 1.9 and 2.6-fold increases in 1.5 and 2.5 mmol/L treatment groups (P<0.01) , whereas the expressions of p-ERK1/2 significantly decreased by 40% and 50% (P<0.01) . No significant change was observed in p-JNK protein expression between the chloroacetic acid-treated and control groups. In comparison with the vehicle control and the exposed group, p-p38, p-ERK1/2, p-JNK protein expressions significantly declined in the inhibitor controls and inhibitor groups. Compared with the controls, the cell survival rates had significant reductions of 28%, 18%, 36% and 26% respectively in chloroacetic acid treated group, SB203580 group, U0126 group and SP600125 group, and the apoptosis rates in the abovementioned groups were 7, 4, 8 and 7 times. Compared with chloroacetic acid-treated group, the cell viability increased by 14% in SB203580 group and decreased by 11% in U0126 group, and the cell apoptosis rates decreased by 36% in SB203580 group and increased by 18% in U0126 group (P<0.05) . But no significant changes were observed in cell viability and apoptosis between SP600125 and chloroacetic acid-treated group. Conclusion Chloroacetic acid might activate p38 MAPK signaling pathway and inhibit ERK1/2 MAPK signaling pathway. The signaling pathways of p38 and ERK1/2 MAPK are involved in 16HBE cell apoptosis induced by chloroacetic acid, but JNK is not involved in chloroacetic acid-induced 16HBE cell apoptosis.

OBJECTIVE: To investigate the effect of toluene diisocyanate(TDI) on the activation of autophagy and expression of inflammatory cytokines interleukin(IL)-4 and IL-6 in normal human bronchial epithelial cells(16 HBE). METHODS: i) We prepared TDI-human serum albumin(HSA) and determined the mass concentration of TDI in TDI-HSA. ii) The cells were treated with TDI-HSA and HSA at concentrations of 0.00-400.00 mg/L for 12 hours. CCK-8 assay was used to determinate the cell viability, and TDI-HSA and HSA doses were selected for subsequent experiments. iii) The cells were treated with TDI-HSA and HSA at doses of 0.00-120.00 mg/L for 12 hours, and the levels of reactive oxygen species(ROS) in the cells were detected by flow cytometry. The levels of IL-4 and IL-6 in the cell supernatant were measured by enzyme-linked immunosorbent assay. iv) The cells were treated with TDI-HSA at doses of 0.00-120.00 mg/L for 12 hours, and the autophagy activity was observed under transmission electron microscope. Western blot was utilized to detect the expression of Beclin1, microtubule-associated protein 1 light chain(LC3β) and P62. RESULTS: i) The mass concentrations of TDI in 40.00, 80.00 and 120.00 mg/L TDI-HSA groups were 0.44, 0.89 and 1.33 mg/L respectively. ii) The results of CCK-8 showed that TDI-HSA and HSA at doses below 120.00 mg/L did not affect cell viability, and 0.00-120.00 mg/L was selected as the TDI-HSA and HSA treatment doses for subsequent experiments. iii) The level of ROS in cells and the levels of IL-4 and IL-6 in the supernatant of 16 HBE cells in the TDI-HSA group at 40.00, 80.00, and 120.00 mg/L were higher than that in HSA group at the same dose(P<0.01). The level of ROS in cells and the levels of IL-4 and IL-6 in the supernatant of 16 HBE cells increased with the increase of TDI-HSA doses(P<0.01). iv) Transmission electron microscopy showed that the number of autophagic lysosomes in 16 HBE cells increased significantly, and the number of mitochondrial vacuoles increased in 40.00, 80.00, 120.00 mg/L TDI-HSA group compared with 0.00 mg/L group. With the increase of TDI-HSA dose, the relative expression of Beclin1 protein and LC3β-Ⅱ/Ⅰ ratio in 16 HBE cell supernatant increased(P<0.05), and the relative expression of P62 protein decreased(P<0.05). CONCLUSION: TDI-HSA induces increased expression of ROS and inflammatory factors and induces autophagy activation in 16 HBE cells. Autophagy may be an important factor for the development of airway inflammation in TDI-induced occupational asthma.

Objective:To investigate the interventional effect of metformin on pulmonary inflammation and pulmonary fibrosis in silicotic rats.Methods:In April 2019, 48 Wistar male rats of SPF grade were randomly divided into negative control group, metformin control group, silicon dioxide (SiO 2) model group, low, medium and high dose metformin intervention group according to the random number table method, 8 rats in each group. The SiO 2 model group and the low, medium and high dose metformin intervention groups were given 1 ml 50 mg/ml of SiO 2 by intratracheal instillation, the negative control group and the metformin control group were given 1 ml normal saline by intratracheal instillation. 24 hours later, the low, medium and high dose metformin intervention groups and the metformin control group were treated with 100, 200, 400 and 400 mg/kg metformin daily, the control and SiO 2 model groups received normal saline daily. Then the rats were sacrificed at the 28th day after SiO 2 exposure. The changes of rat body weight and pathological examination of rat lung tissue were observed, and the lung organ coefficient, the content of hydroxyproline (HYP) , the expression levels of inflammatory factors transforming growth factor beta1 (TGF-β1) , tumor necrosis factor-alpha (TNF-α) , interleukin-1beta (IL-1β) and the protein expression of E-cadherin (E-Cad) , Vimentin, α-SMA were detected. Results:Compared with the negative control group, SiO 2 model group had a significant decrease in the body weight of rats ( P<0.05) , lung organ coefficient, alveolitis and fibrosis scores, HYP content and the levels of TGF-β1, TNF-α, IL-1β were all significantly increased ( P<0.05) . Compared with the SiO 2 model group, the weights of the rats in the medium and high dose intervention group of metformin increased significantly ( P<0.05) . And after intervention with different doses of metformin, the lung organ coefficient, alveolitis and fibrosis scores, HYP content and the levels of TGF-β1, TNF-α and IL-1β were significantly decreased ( P<0.05) . Immunohistochemistry and Western blotting results showed that compared with the negative control group, the expression of E-Cad of the SiO 2 model group was decreased, and the expression levels of Vimentin and α-SMA were significantly increased ( P<0.05) . After metformin intervention, the expression of E-Cad was significantly increased, the expression levels of Vimentin and α-SMA were significantly decreased ( P<0.05) . Conclusion:Metformin can reduce lung tissue inflammation and fibrosis in rats exposed to SiO 2 dust, which may be related to reducing the expression of inflammatory factors in lung tissue and inhibiting the EMT process.

Objective To explore the effect of sling exercise with Tuina therapy on kinesiophobia in old patients with lumbar disc herniation,and analyze the mechanism based on brain-bone axis. Methods A total of 56 old patients with chronic lumbar disc herniation and kinesiophobia were selected from the Reha-bilitation Hospital of the Second Affiliated Hospital of Shandong University of Traditional Chinese Medicine from September,2022 to December,2023;and randomly divided into control group(n=28)and experimental group(n=28).The control group accepted conventional exercise therapy,while the experimental group accepted sling exercise with Tuina therapy,for four weeks.They were assessed with simplified Chinese version of Tampa Scale of Kinesiophobia(TSK),Japanese Orthopaedic Association score(JOA)and Visual Analogue Scale for pain(VAS)before and after treatment,while the bone mineral density(BMD)was tested,the levels of osteoprote-gerin(OPG),norepinephrine(NE)and corticosteroids(Cor)in serum were measured,and the median frequency(MF)of weak-link erector spinae was detected with surface electromyography. Results Two cases dropped off in the control group,and one in the experimental group.The scores of all the assessment improved in both groups after treatment(|t|>14.168,P<0.001),as well as the serum levels of OPG,NE and Cor(|t|>2.103,P<0.05),BMD(|t|>2.726,P<0.05),and MF of erector spinae(|t|>14.736,P<0.001);all of them were better in the experimental group than in the control group(|t|>2.154,P<0.05). Conclusion Sling exercise with Tuina therapy can improve the pain and kinesiophobia of lumbar disc herniation in the old adults,which may promote the recovery of physical and mental function through regulating the levels of hor-mones and neurotransmitters related to the brain-bone axis.

Objective:To investigate the interventional effect of metformin on pulmonary inflammation and pulmonary fibrosis in silicotic rats.Methods:In April 2019, 48 Wistar male rats of SPF grade were randomly divided into negative control group, metformin control group, silicon dioxide (SiO 2) model group, low, medium and high dose metformin intervention group according to the random number table method, 8 rats in each group. The SiO 2 model group and the low, medium and high dose metformin intervention groups were given 1 ml 50 mg/ml of SiO 2 by intratracheal instillation, the negative control group and the metformin control group were given 1 ml normal saline by intratracheal instillation. 24 hours later, the low, medium and high dose metformin intervention groups and the metformin control group were treated with 100, 200, 400 and 400 mg/kg metformin daily, the control and SiO 2 model groups received normal saline daily. Then the rats were sacrificed at the 28th day after SiO 2 exposure. The changes of rat body weight and pathological examination of rat lung tissue were observed, and the lung organ coefficient, the content of hydroxyproline (HYP) , the expression levels of inflammatory factors transforming growth factor beta1 (TGF-β1) , tumor necrosis factor-alpha (TNF-α) , interleukin-1beta (IL-1β) and the protein expression of E-cadherin (E-Cad) , Vimentin, α-SMA were detected. Results:Compared with the negative control group, SiO 2 model group had a significant decrease in the body weight of rats ( P<0.05) , lung organ coefficient, alveolitis and fibrosis scores, HYP content and the levels of TGF-β1, TNF-α, IL-1β were all significantly increased ( P<0.05) . Compared with the SiO 2 model group, the weights of the rats in the medium and high dose intervention group of metformin increased significantly ( P<0.05) . And after intervention with different doses of metformin, the lung organ coefficient, alveolitis and fibrosis scores, HYP content and the levels of TGF-β1, TNF-α and IL-1β were significantly decreased ( P<0.05) . Immunohistochemistry and Western blotting results showed that compared with the negative control group, the expression of E-Cad of the SiO 2 model group was decreased, and the expression levels of Vimentin and α-SMA were significantly increased ( P<0.05) . After metformin intervention, the expression of E-Cad was significantly increased, the expression levels of Vimentin and α-SMA were significantly decreased ( P<0.05) . Conclusion:Metformin can reduce lung tissue inflammation and fibrosis in rats exposed to SiO 2 dust, which may be related to reducing the expression of inflammatory factors in lung tissue and inhibiting the EMT process.

OBJECTIVE: To explore the effect of reactive oxygen species(ROS) and cell cycle in bone marrow cells in benzene-induced aplastic anemia(AA) mouse model. METHODS: Specific pathogens free male CD1 mice were randomly divided into control group and exposure group(n=10, each group). The mice in exposure group were subcutaneously injected with benzene at a dose of 2 mL/kg body weigh diluted 1 ∶1 in corn oil, while the mice in control group were treated with equal volume of corn oil, 3 times a week for a total of 25 times. After exposure, the blood routine and reticulocyte percentage of peripheral blood of mice were examined. The femur histopathology was performed. The levels of benzene and its metabolites hydroquinone, and phenol in blood, liver and bone marrow were tested by solid-phase extraction gas chromatography mass spectrometry. The level of ROS and the changes of cell cycle in bone marrow mononuclear cells(BMMNCs) were determined by flow cytometry. The protein expression of Cyclin D1 and cyclin-dependent kinase 4(CDK4) in BMMNCs was detected by Western blot. RESULTS: Since the 10 th benzene exposure, the body mass of mice in the exposure group was lower than that in the control group at the same time point(P<0.05). After the benzene exposure, all the counts of white blood cell, red blood cell, platelet, and hemoglobin level and reticulocyte percentage in peripheral blood of mice in the exposure group were decreased when compared with the control group(P<0.05). Bone marrow histopathological examination showed that bone marrow hematopoietic cells were decreased and non-hematopoietic cells were increased in the exposure group. In this study, a mouse model of AA induced by benzene was successfully established. The levels of benzene, hydroquinone and phenol in exposure group increased in blood, liver, and bone marrow compared to control group(P<0.05). Furthermore, the level of benzene from high to low were blood, liver and bone marrow, while the levels of hydroquinone and phenol were mainly stored in the blood and bone marrow in exposure group. Compared with the control group, the level of ROS, S phase fraction, and the relative protein expression of Cyclin D1 and CDK4 in BMMNCs increased, while the G1 phase fraction decreased in exposure group(P<0.01). CONCLUSION: The results suggest that benzene and its metabolites induce an increase of ROS level and S phase cell arrest, that play an important role in the pathogenesis and development of benzene-induced AA.

Objective: To investigate the role of microRNA-29b-3p (miRNA-29b-3p) and miRNA-34c-3p in the process of pulmonary fibrosis, we detected the expression levels of miRNA-29b-3p and miRNA-34c-3p in the lung tissue of rats exposed to silica and A549 cells. Methods: SPF male Wistar rats were randomly divided into 1, 7, 14, 21, 28 d control group and silica (SiO₂) dusting group, with 6 rats in each group. One-time non-exposure method was used to infuse 1ml SiO₂ suspension. The rat SiO₂ dusting group was established in the liquid, and the control rats were intratracheally injected with 1 ml of sterile physiological saline in the same manner. The lung tissues of each group were collected at the corresponding time points after dusting. Three of the rats were taken out for pathological observation, and the other three were used to screen differentially expressed miRNAs in lung tissue by miRNA microarray technology. A549 cells were cultured at the in vitro cell level and divided into control group, SiO₂ stimulation group and TGF-β₁ stimulation group, and cells were collected at 12, 24 and 48 h after treatment. The expression levels of miRNA-29b-3p and miRNA-34c-3p in rat lung tissue and A549 cells were verified by real-time PCR (qRT-PCR), target gene prediction of miRNA-29b-3p and miRNA-34c-3p and perform GO enrichment analysis and KEGG pathway analysis. Results: The weight growth rate of the control group was significantly higher than that of the SiO₂ dusting group. Compared with the control group, the lung mass and lung coefficient of the SiO₂ dusting group were significantly increased (P<0.05). The inflammatory response of the lungs in the control group was significantly reduced at 21 and 28 days, and the inflammatory cells infiltrated in the lung tissue of the SiO2 group. The rats in the control group had a small amount of collagen at 21 and 28 days. A large amount of collagen fiber deposition began to appear in the lung tissue of rats exposed to SiO₂ for 21 days. Compared with the control group, the expression levels of miRNA-29b-3p and miRNA-34c-3p in the SiO₂ dusting group were significantly down-regulated, and there was significant difference compared with the control group (P<0.05). The expression levels of miRNA-29b-3p and miRNA-34c-3p in A549 cells treated with SiO₂ and human recombinant TGF-β1 were significantly lower than those in the control group at 24 h and 48 h, and the difference was statistically significant (P<0.05). Conclusion Down-regulation of miRNA-29b-3p and miR-34c-3p in rat lung tissue A549 cells may be associated with the development of early silicosis and is expected to be an indicator of early silicosis diagnosis and prognosis.