Tim-3 facilitates immune escape in benzene-induced acute myeloid leukemia mouse model by promoting macrophage M2 polarization.

Benzene poisoning can cause acute myeloid leukemia (AML) through a variety of passways. Tim-3 has gained prominence as a potential candidate in mediating immunosuppression in tumor microenvironments. The macrophage polarization is also related to immune escape. Herein, we reported that Tim-3 and macrophage M2 polarization play a vital role in benzene-induced AML. First, the benzene-induced AML C3H/He mouse model was constructed by subcutaneously injecting 250 mg/kg of benzene. After six months, macrophage phenotype, cytokines, and Tim-3 expression levels were investigated. Flow cytometry assay revealed that the T-cell inhibitory receptor Tim-3 was significantly upregulated in both bone marrow and spleen of the benzene-induced AML mouse model. Elisa's results displayed a decreased serum level of IL-12 while increased TGF-ß1. Mechanistically, changes in cytokine secretion promote the growth of M2-type macrophages in the bone marrow and spleen, as determined by immunofluorescence assay. The increased levels of PI3K, AKT, and mTOR in the benzene-exposure group further proved the crucial role of Tim-3 in regulating the functional status of macrophages in the AML microenvironment. These results demonstrate that Tim-3 and macrophage polarization may play a vital role during the immune escape of the benzene-induced AML. This study provides a new potential intervention site for immune checkpoint-based AML therapeutic strategy.

Effects of tacrolimus on the TGF­ß1/SMAD signaling pathway in paraquat­exposed rat alveolar type II epithelial cells.

Paraquat is a highly toxic pesticide, which often causes pulmonary interstitial fibrosis after poisoning, and there is no specific antidote. At present, limited studies have reported that tacrolimus, as an immunosuppressant, can inhibit pulmonary fibrosis, but the specific mechanism remains unknown. The aim of the present study was to demonstrate the effect of tacrolimus on the TGF­ß1 pathway associated with pulmonary fibrosis in paraquat exposed alveolar type II epithelial cells, and to identify the antipulmonary fibrosis mechanism of tacrolimus The rat alveolar epithelial type II RLE­6TN cell line was exposed to paraquat and treated with or without tacrolimus for 24 h, or with a TGF­ß1 receptor type I/II inhibitor (LY2109761) for 1, 4, 8 or 16 h. MTT assays were used to detect the viability of rat alveolar type II epithelial cells under these different treatment conditions, while the concentrations of TGF­ß1, SMAD3, SMAD7 and connective tissue growth factor (CTGF) in the cell culture supernatant were determined using ELISAs. Additionally, reverse transcription­quantitative PCR and immunofluorescence were used to analyze the mRNA and protein expression levels of TGF­ß1, SMAD3, CTGF and SMAD7. The results demonstrated that the inhibition of the proliferation of RLE­6TN cells exposed to 200 nmol/l paraquat was 26.05±2.99%. The inhibition rate of 10 ng/ml tacrolimus on paraquat­exposed alveolar type II epithelial cells was 18.40±3.49%. The inhibition rate caused by 5 µmol/l LY2109761 was 26.56±4.49%. The expression levels of TGF­ß1, SMAD3 and CTGF, as well as their concentrations in the culture supernatant, were significantly downregulated in the tacrolimus group compared with the paraquat group. However, both the concentration and expression levels of SMAD7 were significantly upregulated in the tacrolimus group compared with the paraquat group. In conclusion, tacrolimus can reduce the levels of TGF­ß1, SMAD3 and CTGF, increase the level of SMAD7 in TGF­ß1 signaling pathway and protect the development of pulmonary fibrosis in paraquat exposed alveolar epithelial cells.

[The experimental study of therapeutical effects of KANGFUXINYE on upper gastrointestinal injury induced by paraquat in rats].

OBJECTIVE: To investigate the therapeutical effects of KANGFUXINYE on the upper gastrointestinal injury induced by paraquat in rats, and to explore the proper mechanism. METHODS: A total of 120 adult Wistar male rats were randomly divided into three groups, control group (CG), model group (MG) and treatment group (TG), 40 rats each group. The MG and TG were given 20% paraquat 50 mg/kg by oral administration, after 2 h the TG was given KANGFUXINYE solution 1.5 ml by oral administration, 3 times a day. The CG was given normal saline. On the 3rd, 6th, 9th, 12th and 15th days after exposure, 8 rats of each group were killed respectively, and the tissues from esophagus and stomach were collected and examined by HE staining for observing the mucosa injury. The superoxide dismutase (SOD) and malondialdehyde (MDA) of serum were detected. RESULTS: On the 3rd, 6th, 9th, 12th and 15th days after exposure, the results of pathological examination showed that the mucosa injury in TG was significantly relieved as compared with MG, the activity of serum SOD reduced obviously and the MDA levels increased significantly in MG, as compared with CG (P<0.05). The activity of serum SOD increased obviously and the MDA levels decreased significantly in TG, as compared with MG (P<0.05). CONCLUSION: The results of present indicate that KANGFUXINYE has the therapeutical effects on the upper gastrointestinal injury caused by paraquat in rats. The mechanism of therapeutical effects may be due to the increasing SOD activity, eliminating free radicles and inhibiting the lipid peroxidation.

[Experimental therapy of penehyclidine hydrochloride on paraquat-induced acute lung injury].

OBJECTIVE: To observe the therapeutic effect and mechanism of penehyclidine hydrochloride on paraquat-induced acute lung injury. METHODS: 80 healthy adult male Wistar rats were randomly assigned into control groups (10 rats), 100 mg/kg PQ group (10 rats), 100 mg/kg PQ plus 33 µg/kg penehyclidine hydrochloride treatment group (30 rats), 100 mg/kg PQ plus 66 µg/kg penehyclidine hydrochloride treatment group (30 rats). The two treatment groups were executed respectively at 36 h, 72 h and 7 d. Lung tissues were used to assess histopathological change by HE staining. The level of MMP-2, caveolin-1 and HYP were detected in the lung homogenate. The serum and BALF contents of ET were measured. RESULTS: Pathology inspection confirmed that the model of acute rat pulmonary injury were duplicated successfully. The level of MMP-2, HYP in lung tissues and the serum and BALF ET contents in PQ group were (1.77 ± 0.40) µg/g, (2.91 ± 0.79) µg/g, (505.23 ± 124.69) µg/ml, (640.38 ± 136.60) µg/ml. The level of those was higher than that in control group [(0.95 ± 0.66) µg/g, (1.48 ± 0.69) µg/g, (95.48 ± 46.01) µg/ml, (200.40 ± 88.39) µg/ml, P < 0.05]; The above-mentioned index in two treatment groups was lower than that in PQ group (P < 0.05). The caveolin-1 content [(1.77 ± 0.82) µg/g] in PQ group was lower than that in control group [(5.39 ± 1.68) µg/g, P < 0.05]. The level of caveolin-1 in two treatment groups was higher than that in PQ group (P < 0.05). CONCLUSION: Penehyclidine hydrochloride can decrease the level of MMP-2, HYP in lung tissues and the ET in serum and BALF, increase that of caveolin-1 and lessen the damage induced by paraquat.

[Clinical observation of rheumatoid arthritis associated interstitial lung disease patients and changes of serum cytokines thereof].

OBJECTIVE: To observe the clinical feature of rheumatoid arthritis associated interstitial lung disease (RA-ILD) patients and changes of serum cytokines tumor growth factor (TGF)-beta 1, tumor necrosis factor (TNF)-alpha, insulin-like growth factor (IGF)-1, and platelet derived growth factor (PDGF)-AB. METHODS: The clinical manifestations, lung high resolution CT (HRCT), lung functions, blood gas and other relative laboratory findings of 30 RA-ILD patients and 35 RA patients were observed. ELISA was used to detect the levels of TGF-beta 1, TNF-alpha, IGF-1, and PDGF-AB. Thirty healthy volunteers were observed too as controls. RESULTS: The clinical manifestations of RA-ILD patients were more serious than those of the RA patients. The ESR was faster, the serum C-reactive protein, rheumatoid factor (RF), and globulin levels higher, and pulmonary arterial pressure higher too in the RA-ILD patients than in the RA patients (all P<0.01). The main respiratory manifestations of the RA-ILD patients were cough, expectoration, chest distress, short breath, chest pain, change of breath sounds, Velcro râles, and dyspnea. The main lung HRCT findings included thickening of interlobular septum and bronchial wall, pachynsis pleurae, mosaic sign, bronchiectasis, emphysema, patching shadow, honeycombing, fibrous scar, etc. Pulmonary function test showed that the levels of vital capacity, forced vital capacity, maximum midexpiratory flow, and diffusing capacity of the lung for carbon monoxide of the RA-ILD patients were all significantly lower than those of the RA patients (all P<0.01). Arterial gas test showed that the PO2 of the RA-ILD patients was significantly lower than that of the RA patients (P<0.01). The TGF-beta 1; TNF-alpha, IGF-1, and PDGF-AB of both the RA-ILD and RA patients were all significantly higher than those of the healthy volunteers (all P<0.01), and the levels of these cytokines of the RA-ILD patients were all higher than those of the RA patients (all P<0.01). CONCLUSION: The symptoms and signs of the RA-ILD patients are more serious, the lung HRCT changes more obvious, lung function decreases, and the levels of TGF-beta 1, TNF-alpha, IGF-1, and PDGF-AB increase.

[Clinical study on treatment of acute paraquat poisoning].

OBJECTIVE: To investigate the clinical therapeutic effect of methylprednisolone combined with cyclophosphamide and Etanercept method on acute paraquat poisoning. METHODS: 136 patients with acute paraquat poisoning were divided into the normal therapy group and the intensive therapy group randomly. Methylprednisolone, cyclophosphamide and Etanercept were used in the intensive therapy group. Methylprednisolone 500 mg was given intravenously per day for continuous three days followed by 200 mg intravenous per day. Then methylprednisolone was decreased gradually 14 d or 21 d later according to the patient's condition. Cyclophosphamide 600 mg was given intravenously twice weekly for 2 weeks and Etanercept 25 mg was given hypodermic injection twice weekly for 3 weeks. Curative effect evaluation was done at 7, 14, 21 d and 12 weeks after therapy. RESULTS: The survival rate of the intensive therapy group was obviously higher than that of the normal therapy group (P<0.01) on 7, 4, 21 d and 12 weeks. The cure rate of the intensive group were 94.6% (intake dose<50 ml 20% paraquat solution), 75.0% (intake dose 50 approximately 100 ml 20% paraquat solution), 12.5% (intake dose>100 ml 20% paraquat solution) respectively, while the cure rate of the normal group were 16.7% (intake dose<50 ml 20% paraquat solution), 8.3% (intake dose 50 approximately 100 ml 20% paraquat solution), 0% (intake dose>100 ml 20% paraquat solution) respectively. The total cure rate of the intensive therapy group (78.3%) 12 weeks later was higher than that of the normal group (11.9%). CONCLUSION: Methylprednisolone combined with cyclophosphamide and Etanercept intensive therapy has the curative effect on acute paraquat poisoning.