OBJECTIVE To investigate the effect and mechanism of bumetanide on lung injury in chronic obstructive pulmonary disease （COPD） model rats. METHODS COPD rat model was induced by lipopolysaccharide， and they were randomly divided into model group （COPD group）， bumetanide low-dose and high-dose groups （Bumetanide-L group， Bumetanide-H group）， bumetanide high-dose+Yes-associated protein/transcriptional coactivator containing PDZ-binding motif （YAP/TAZ） signaling pathway activator group （Bumetanide-H+PY-60 group）， with 12 rats in each group. Another 12 normal rats were selected as normal control group （Control group）. Thirty minutes before modeling， bumetanide/normal saline was inhaled or/and PY-60/ normal saline was injected into the tail vein. On the next day after the completion of modeling and drug administration， the pulmonary function index of the rats in each group was measured [forced expiratory volume in 0.3 seconds （FEV0.3）， forced vital capacity （FVC）， peak expiratory flow （PEF）， FEV0.3/FVC]. The levels of tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6） and IL-1β in bronchoalveolar lavage fluid （BALF） were determined； the pathological morphology of lung tissue and degree of pulmonary fibrosis were observed. The expression levels of transforming growth factor- β （TGF- β）， α -smooth muscle actin （α-SMA） and TAZ protein as well as the phosphorylation of YAP protein in lung tissues were detected. RESULTS Compared with COPD group， the pathological injury of lung tissue in Bumetanide-L and Bumetanide-H groups was alleviated； the exfoliation of lung epithelial cells， tube wall thickening and the degree of pulmonary fibrosis were alleviated； inflammatory cell infiltration was reduced， and blue collagen deposition was reduced； FEV0.3， FVC， FEV0.3/FVC and PEF were significantly increased， while the lung injury score， levels of TNF-α， IL-6， IL-1β， expression levels of TGF-β， α-SMA and TAZ protein and the phosphorylation of YAP protein were significantly decreased （P＜0.05）. PY-60 could significantly reverse the improvement effects of bumetanide on above indexes （P＜0.05）. CONCLUSIONS Bumetanide can alleviate lung injury， inflammatory response and pulmonary fibrosis in COPD rats， and its mechanism is related to inhibiting YAP/TAZ signaling pathway.

OBJECTIVE To investigate the effect and mechanism of Jingangteng capsules in the treatment of non-alcoholic fatty liver disease （NAFLD）. METHODS Thirty-two SD rats were randomly divided into normal group and modeling group. The modeling group was fed a high-fat diet to establish a NAFLD model. The successfully modeled rats were then randomly divided into model group， atorvastatin group[positive control， 2 mg/（kg·d）]， and Jingangteng capsules low- and high-dose groups [0.63 and 2.52 mg/（kg·d）]， with 6 rats in each group. The pathological changes of the liver were observed by hematoxylin-eosin staining and oil red O staining. Enzyme-linked immunosorbent assay was performed to determine the serum levels of triglycerides （TG）， total cholesterol （TC）， high-density lipoprotein cholesterol （HDL-C）， low-density lipoprotein cholesterol （LDL-C）， alanine transaminase （ALT）， aspartate transaminase （AST）， tumour necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， IL-6， IL-18. 16S rDNA amplicon sequencing and metabolomics techniques were applied to explore the effects of Jingangteng capsules on gut microbiota and metabolisms in NAFLD rats. Based on the E-mail：591146765@qq.com metabolomics results， Western blot analysis was performed to detect proteins related to the nuclear factor kappa-B （NF-κB）/NOD-like receptor family protein 3 （NLRP3） signaling pathway in the livers of NAFLD rats. RESULTS The experimental results showed that Jingangteng capsules could significantly reduce the serum levels of TG， TC， LDL-C， AST， ALT， TNF-α， IL-1β， IL-6， IL-18， while increased the level of HDL-C， and alleviated the hepatic cellular steatosis and inflammatory infiltration in NAFLD rats. They could regulate the gut microbiota disorders in NAFLD rats， significantly increased the relative abundance of Romboutsia and Oscillospira， and significantly decreased the relative abundance of Blautia （P＜0.05）. They also regulated metabolic disorders primarily by affecting secondary bile acid biosynthesis， fatty acid degradation， O-antigen nucleotide sugar biosynthesis， etc. Results of Western blot assay showed that they significantly reduced the phosphorylation levels of NF-κB p65 and NF-κB inhibitor α， and the protein expression levels of NLRP3， caspase-1 and ASC （P＜0.05 or P＜0.01）. CONCLUSIONS Jingangteng capsules could improve inflammation， lipid accumulation and liver injury in NAFLD rats， regulate the disorders of gut microbiota and metabolisms， and inhibit NF-κB/NLRP3 signaling pathway. Their therapeutic effects against NAFLD are mediated through the inhibition of the NF-κB/NLRP3 signaling pathway.

ObjectiveTo explore the role of lead exposure in the phenotypic transformation of vascular smooth muscle cells (VSMC), and to provide new insights for the mechanism of lead impact on vascular lesions. MethodsMouse aortic smooth muscle cells (MOVAS) were divided into a control group (0 μmol·L^-1), low concentration lead groups (0.1, 1, 5, and 10 μmol·L^-1), and high concentration lead groups (15, 25, and50 μmol·L^-1). MTT assays were used to assess the proliferation of the cells, and scratch assays were implicated to measure migration ability of the cells. Fluorescence quantitative PCR was employed to determine levels of mRNA expression for smooth muscle actin α (α⁃SMA), smooth muscle 22 alpha (SM22α), synthetic phenotype-related genes osteopontin (OPN), matrix metalloproteinase 9 (MMP9), and the transcription factor SOX9. Immunoblotting was used to determine levels of protein expression for α-SMA, OPN, and MMP9. ResultsProliferation of MOVAS was observed under the lead ions concentrations of 0‒50 µmol·L^-1, with a significant increase of proliferation compared to the control group at the concentrations of 5‒50 µmol·L^-1 (all P<0.05). The migration ability of cells gradually increased at the concentrations of 0‒10 µmol·L^-1, with a significant increase at 5 (q=4.574, P=0.003) and 10 µmol·L^-1 (q=10.570, P<0.001) compared to the control group. The 10 µmol·L^-1 lead ions significantly reduced the levels of mRNA expression for vascular smooth muscle contractile phenotype genes α⁃SMA (q=7.426, P<0.001) and SM22α (q=4.766, P=0.001), while significantly increasing the levels of mRNA expression for OPN (q=11.330, P<0.001), MMP9 (q=7.842, P<0.001), and SOX9 (q=11.120, P<0.001) genes. Furthermore, the 10 µmol·L^-1 lead ions significantly reduced the levels of protein expression for the vascular smooth muscle contractile phenotype marker α-SMA protein (q=2.897, P=0.049), while significantly increasing the levels of protein expression for the synthetic markers OPN (q=3.188, P=0.031) and MMP9 (q=3.292, P=0.026), compared to the control group. ConclusionTreatment with lead in vitro induced VSMC to differentiate from contractile phenotype to synthetic phenotype, indicating that a certain dose of lead exposure might be detrimental to the cardiovascular system.

Objective To identify the independent predictors of programmed death-1 (PD-1) inhibitor-related endocrine adverse events and construct a clinically usable risk prediction model. Methods A total of 302 patients with solid tumors treated with PD-1 inhibitors were retrospectively enrolled. According to the presence or absence of endocrine immune-related adverse events (irAEs), the patients were divided into case group and control group. The clinical and laboratory indexes were compared between the two groups. Multivariable logistic regression was used to confirm independent predictors of endocrine irAEs. The nomogram was constructed, while the receiver operating characteristic (ROC) curve was used to test the prediction performance of the model. Results The overall incidence of endocrine irAEs was 21.9% (66/302), and the incidence of hypothyroidism was 19.5% (59/302). The age, PD-1 inhibitors, free thyroxine, thyroid peroxidase antibody (TPOAb), thyroglobulin, amylase, lymphocyte subset CD3 expression were statistically different between the two groups (P＜0.05). Multivariable logistic regression showed that higher expression of lymphocyte subset CD3 was a protective factor to prevent endocrine irAEs occurrence (P＝0.004), while age＜60 years, higher TPOAb and use of pembrolizumab were independent risk factors of endocrine irAEs (P＜0.05). The nomogram model thus constructed, and when the threshold probability of the model exceeded 0.1, its net benefit was higher. ROC curve showed that the AUC of the model to predict endocrine irAEs was 0.760. The prediction result of the model was highly consistent with the actual result. Conclusions The age, type of PD-1 inhibitor, baseline TPOAb level, and baseline CD3 expression can independently predict endocrine irAEs occurrence or not. The nomogram model based on this model has good predictive efficiency, which can provide reference for early identification of high-risk patients and immunotherapy management.

Objective To establish a cell bank of extrahepatic cholangiocarcinoma (ECC)-derived organoids and investigate the key factors influencing the organoids generation. Methods The tumor samples from patients with portal cholangiocarcinoma (pCCA) and distal cholangiocarcinoma (dCCA) were used to isolate cells, and these cells were cultured using three-dimensional (3D) technique to establish ECC organoids. Histological characteristics of the organoids were evaluated and identified through hematoxylin-eosin (HE) and immunohistochemistry stainings. The success rates of organoids generation from different tumor types were compared. And clinical characteristics of patients between successful and failure culture groups were compared. Results The success rates of organoids establishment from pCCA and dCCA were all low, with 42.4% (14/33), 51.9% (14/27), respectively. The tumor was larger in successful group than that in failure group (P＜0.001); there was no statistical difference in tumor differentiation status, microvascular invasion, and perineural invasion between the two groups. Conclusions The successful rate of ECC-derived organoids establishment is low, and larger tumor has higher successful culture rate.

Objective To explore the predictive value of two models based on machine learning algorithms in predicting the initial and subsequent doses of tacrolimus in kidney transplant recipients. Methods A retrospective analysis was conducted on the medical records of 1 013 Chinese kidney transplant recipients at the First Affiliated Hospital of Sun Yat-sen University from January 2015 to April 2019, focusing on the initial and subsequent doses in kidney transplant recipients. Thirty-three variables were collected for the initial dose, and twenty-six variables for the subsequent dose. A genetic algorithm combined with a random-restart hill-climbing algorithm was used to determine a small number of key clinical variables through majority voting, and variables with Lasso regression coefficients less than the optimal variable coefficient threshold were further eliminated. The selected clinical variables were input into a cascaded deep forest (CDF) and TabNet deep neural network for analysis and comparison based on structured tabular data, and the leave-one-subject-out method was used for validation. Results A total of 613 recipients were included in the training set, and 116 recipients were in the external validation set. In the initial dose algorithm of tacrolimus, the clinical variables ultimately included target concentration, time from surgery to target concentration, body weight, gender, type of surgery, time from surgery to first dose, WuZhi capsule, calcium channel blocker, creatinine, hemoglobin and CYP3A5. In the subsequent dose algorithm, the clinical variables ultimately included target concentration, time from surgery to target concentration, WuZhi capsule, creatinine, alanine aminotransferase, aspartate aminotransferase, previous dose, previous dose concentration and time from surgery to previous concentration. Based on the above variables, the TabNet model showed better predictive performance than the CDF model: in the initial dose prediction, the accuracy of the predicted dose within ±20% of the actual dose was 0.801, and the fitting index R² was 0.436; in the subsequent dose prediction, the corresponding accuracy and R² were 0.939 and 0.902, respectively. The results of feature contribution showed that CYP3A5 and target concentration contributed the most to the prediction of initial dose, while previous dose and its corresponding concentration had the greatest impact on subsequent dose prediction. In addition, the results of independent external validation were also satisfactory. Conclusions The optimized TabNet predictive model may provide important reference for drug dose prediction based on machine learning algorithms in clinical practice.

OBJECTIVE To investigate the effect of Jiawei Tianwang Buxin Dan(JWBXD)on insomnia in rats exposed to simulated high-altitude conditions.METHODS ① Thirty SD rats were randomly divided into the normal control,model,model+Jiawei Tianwang Buxin Dan(JWBXD,9.6 mg·kg-1),model+Tianwang Buxin Dan(TWBXD,9.6 mg·kg-1),and model+diazepam(DZP,3 mg·kg-1)groups.Rats,except for the normal control group,were subjected to a low-pressure,low-oxygen animal experimental chamber simulating a 5000 m altitude.Respective drugs were ig administrated once daily at 9:00 for seven days,and signal acquisition and sleep analysis were conducted by a wireless physiological sig-nal telemetry system.②Forty rats were randomly divided into five groups as described in ①.Through-out the experiment,the general condition and body mass of the rats were observed daily.Drug adminis-tration lasted for seven days,and grip strength was tested one hour after the final administration.ELISA was used to measure the levels of corticotropin-releasing hormone(CRH),adrenocorticotropic hor-mone(ACTH),corticosterone(CORT),and melatonin(MLT)in serum.Western blotting was performed to measure the expression levels of core clock proteins period circadian regulator 2(Per2),circadian locomotor output cycles(Clock),cryptochrome 2(Cry2),brain-muscle arnt-like protein 1(Bmal1),nuclear receptor subfamily 1,group D member 1(NR1D1),glycogen synthase kinase-3β(GSK-3β),as well as acetylserotonin O-methyltransferase(ASMT)in the hypothalamus and pineal gland,respectively.RESULTS ① Compared with the normal control group,the model group exhibited a decrease in total sleep time(P<0.01),an increase in wakefulness(P<0.01),a significant reduction in slow wave sleep(SWS)(P<0.05)and the mean bouts duration(P<0.05).Compared with the model group,both DZP and JWBXD(P<0.01)prolonged sleep time and suppressed wakefulness(P<0.01)in the hypoxic envi-ronment.DZP and JWBXD prolonged SWS(P<0.05,P<0.01),while TWBXD had no significant effect.JWBXD improved the mean bouts duration of SWS in the model rats(P<0.01),whereas no such improvement was observed in model+DZP and model+TWBXD groups.② Compared with the normal control group,the model group showed a significant decrease in forelimb grip strength(P<0.01),increased levels of serum ACTH(P<0.05),CRH,and CORT(P<0.01),and decreased MLT levels(P<0.05).The expression levels of Per2,Cry2,GSK-3β,and NR1D1 in the hypothalamus were downregu-lated(P<0.05,P<0.01),while Bmal1 and Clock were upregulated(P<0.05,P<0.01).ASMT expression in the pineal gland was decreased(P<0.05).Compared with the model group,JWBXD and TWBXD enhanced forelimb grip strength(P<0.01),reduced serum CORT and ACTH levels(P<0.05),decreased CRH levels(P<0.01),and restored MLT levels(P<0.01).JWBXD upregulated the expression levels of Per2,Cry2,GSK-3β and NR1D1 in the hypothalamus(P<0.05,P<0.01),but downregulated Bmal1 and Clock expression(P<0.05,P<0.01).TWBXD downregulated Bmal1 expression in the hypothalamus(P<0.01)and increased NR1D1 expression(P<0.05).DZP significantly enhanced the expression levels of Per2,Cry2 and NR1D1 in the hypothalamus(P<0.01).JWBXD,TWBXD and DZP improved ASMT expression in the pineal gland(P<0.05).CONCLUSION JWBXD can improve sleep structure and prolong the duration of SWS in rats exposed to simulated high-altitude conditions.The mechanisms may involve the regulation of core clock protein expressions in the hypothalamus,promotion of mela-tonin secretion,and inhibition of HPA axis hyperactivity.

OBJECTIVE To investigate whether aldo-keto reductases(AKRs)can act as a nitrore-ductase(NR)and bioactivate aristolochic acid Ⅰ(AA-Ⅰ)to produce AA-Ⅰ-DNA adducts.METHODS① Human-induced hepatocytes(hiHeps)and human bladder RT4 cells were used as tool cells and treated with AA-Ⅰ0,0.5,1.0 and 2 μmol·L-1 for 24 h.Cell viability was detected using the CCK-8 method,and the half maximal inhibition concentration(IC50)was calculated using the CCK-8 method and the level of DNA adduct production was calculated.②hiHeps and RT4 cells were treated with AKR inhibitor luteotin(0,5,10 and 25 μmol·L-1)+AA-Ⅰ 0.2 and 1.0 μmol·L-1 for 24 h,respectively,and the levels of DNA adducts were detected by a liquid chromatography-tandem mass spectrometer(LC-MS/MS).③hiHeps cells were incubated with 80 nmol·L-1 small interfering RNAs(si-AKRs)for 48 h and treated with AA-Ⅰ1.0 μmol·L-1 for 24 h.Real-time qualitative PCR(RT-qPCR)method was used to detect the mRNA expression of AKRs gene and LC-MS/MS technology was used to investigate the effect of specific AKR gene knockdown on DNA adduct levels.④500 nmol·L-1 human AKR recombinant proteins AKR1A1 and AA-Ⅰwere incubated in vitro under anaerobic conditions and the formation of AA-Ⅰ-DNA adducts was detected.RESULTS ①The IC50 of AA-Ⅰto hiHeps and RT4 cells was 1.9 and 0.42 μmol·L-1,respec-tively.The level of DNA adduct production of the two cell lines was significantly different(P<0.01).② Luteolin≥5 μmol·L-1 significantly inhibited the production of AA-Ⅰ-DNA adducts in both cells(P<0.05),and there was a concentration-dependent effect in hiHeps cells(P<0.01,R=0.84).③In the AKR family,the knockdown of AKR1A1 gene up to 80%inhibited the generation of AA-Ⅰ-DNA adducts by 30%-40%.④The AA-Ⅰ-DNA adducts were detected in the incubation of recombinant protein AKR1A1 and AA-Ⅰ under anaerobic conditions in vitro,approximately 1 adduct per 107 nucleotides.CONCLU-SION AKR1A1 is involved in AA-Ⅰ bioactivation,providing a reference for elucidation of the carcino-genic mechanism of AA-Ⅰ.

Objective To explore method of establishing and evaluating an asthma rat model with phlegm and blood stasis syndrome.Methods 60 SD male rats were randomly divided into 5 groups,a normal group,asthma group,combination of disease and syndrome(combination)group,DM group,and KCLW group,with 12 rats in each group.Asthma models were established using ovalbumin(OVA).A syndrome model of phlegm and blood stasis was established using a high-fat diet combined with the ice water bath method.We evaluated the asthma model through animal behavior observation,pathological section observation,inflammation index detection,and airway reactivity measurements.The phlegm and blood stasis syndrome model was evaluated via measurements of rat body mass,blood glucose,blood lipids,coagulation function,and hemorheological indexes and by observing symptoms and syndrome determination by Kechuan Liuwei mixture.Results(1)After OVA induction,the rats in the asthma model group and combination group showed symptoms such as shortness of breath,open mouth breathing,abdominal movement,restlessness,and irritability.HE staining showed the disordered arrangement of the bronchial mucosa in lung tissue,local detachment,thickening of the basement membrane and the bronchial tube wall,narrowing of the lumen,extensive infiltration of inflammatory cells,and congestion of capillaries.Compared with the normal group,the asthma model group and combination group(P＜0.05)had increased serum IL-4,IL-6,and TGF-β1.Penh values were increased after stimulation with various concentrations of Mch(P＜0.05).(2)Rats in the combination group showed symptoms such as chills,curling up with minimal movement,purple and dark claws,purple and black bruises on the tail,loose stools,and unclean perianal area.Compared with the rats in the asthma model group,rats in the combination group had increased body mass(P＜0.05)and blood glucose,triglyceride,and total cholesterol levels(P＜0.05),a shortened thrombin time(P＜0.05),increased fibrinogen content(P＜0.05),and significantly increased whole-blood viscosity at low,medium,and high shear rates(P＜0.05).The indexes were significantly improved after Kechuan Liuwei mixture administration.Conclusions The asthma rat model with phlegm and blood stasis syndrome can be established through OVA induction and high-fat diet combined with ice water bath.The model can be evaluated through behavioral observation,index measurements,and syndrome determination via formulas.

Objective:To compare the effects of different reference brain regions on the semi-quantitative SUV ratio (SUVR) of 18F-Florzolotau PET images of Alzheimer′s disease (AD). Methods:The 18F-Florzolotau PET images of 28 (13 males, 15 females, age (57.3±9.5) years) normal controls (NC), 19 patients (4 males, 15 females, age (73.3±7.3) years) with β-amyloid (Aβ)-positive mild cognitive impairment (MCI) and 40 patients (19 males, 21 females, age (61.9±9.1) years) with AD were collected from Huashan Hospital, Fudan University between November 2018 and July 2020. Six semi-quantitative reference brain regions were defined, including whole cerebellum (WC), cerebellar gray matter (GM), cerebellar white matter (WM), parametric estimation of reference signal intensity (PERSI), WC after partial volume correction (WC_pvc), cerebellar GM after partial volume correction (GM_pvc). SUVR was calculated for 14 ROIs, which included the whole brain defined by the automated anatomical labeling (AAL) template, fusiform, inferior temporal, lingual, middle temporal, occipital, parahippocampal, parietal, posterior cingulate, precuneus defined by the AAL template, and Meta ROI composed of the above brain regions, and braak_Ⅰ-Ⅱ, braak_Ⅲ-Ⅳ, braak_Ⅴ-Ⅵ defined by the Desikan Killiany template. AUC was used to evaluate the classification ability of SUVR, and the correlation between SUVR and clinical scale scores were assessed by Spearman rank correlation analysis. Results:The SUVRs of most brain regions showed a steady upward trend in the AD disease spectrum. In the classification task of NC and MCI, the overall performance of SUVR based on WC_pvc was relatively optimal (AUCs: 0.975-1.000). In the classification task of NC and AD, SUVRs of 10 ROIs based on the WC_pvc method showed the relatively best performance (AUCs: 0.976-1.000). The correlation between SUVR of fusiform based on cerebellar WM and mini-mental state examination (MMSE) score was the strongest ( rs=-0.72, P<0.001), and the SUVR of precuneus based on WC_pvc showed the strongest correlation with clinical dementia rating (CDR) score ( rs=0.78, P<0.001). Conclusion:The SUVR based on WC_pvc method performs well in classification and correlation tasks, and is recommended to be used in semi-quantification of 18F-Florzolotau PET images of AD.