OBJECTIVES: To investigate the role of the BNIP3-PI3K/Akt signaling pathway in mediating the inhibitory effect of Buyang Huanwu Decoction (BYHWT) on mitochondrial autophagy in human synovial fibroblasts from rheumatoid arthritis patients (FLS-RA) cultured under a hypoxic condition. METHODS: Forty normal Wistar rats were randomized into two groups (n=20) for daily gavage of BYHWT or distilled water for 7 days to prepare BYHWT-medicated or control sera. FLS-RA were cultured in routine condition or exposed to hypoxia (10% O₂) for 24 h wigh subsequent treatment with IL-1β, followed by treatment with diluted BYHWT-medicated serum (5%, 10% and 20%) or control serum. AnnexinV-APC/7-AAD double staining and T-AOC kit were used for detecting apoptosis and total antioxidant capacity of the cells, and the changes in ROS, ATP level, mitochondrial membrane potential and Ca²⁺ homeostasis were analyzed. The changes in mRNA and protein expressions of BNIP3, PI3K and AKT and mRNA expressions of LC3, Beclin-1 and P62 were detected using RT-qPCR and Western blotting. RESULTS: Treatment with BYHWT-medicated serum dose-dependently lowered apoptosis rate of IL-1β-induced FLS-RA with hypoxic exposure. The treatment significantly decreased T-AOC concentration, increased ROS production, autophagosome formation and ATPase levels, and lowered mitochondrial membrane potential and Ca²⁺ level in the cells. In IL-1β-induced FLS-RA with hypoxic exposure, treatment with BYHWT-medicated serum significantly increased BNIP3 protein expression, decreased the protein expressions of PI3K and AKT, increased the mRNA expressions of BNIP3 and P62, and lowered the mRNA expressions of PI3K, AKT, LC3 and Beclin-1 without significantly affecting Beclin-1 protein expression. The cells treated with 5% and 10% BYHWT-medicated serum showed no significant changes in LC3 expression. CONCLUSIONS BYHWT inhibits mitochondrial autophagy in IL-1β-induced FLS-RA with hypoxic exposure possibly by inhibiting BNIP3-mediated PI3K/AKT signaling pathway.
Drugs, Chinese Herbal/pharmacology* ; Arthritis, Rheumatoid/pathology* ; Animals ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Autophagy/drug effects* ; Humans ; Fibroblasts/cytology* ; Rats, Wistar ; Membrane Proteins/metabolism* ; Rats ; Phosphatidylinositol 3-Kinases/metabolism* ; Mitochondria/metabolism* ; Cells, Cultured ; Proto-Oncogene Proteins/metabolism* ; Apoptosis/drug effects* ; Cell Hypoxia ; Synovial Membrane/cytology* ; Male ; Mitochondrial Proteins

Objective:This study was conducted to investigate the independent risk factors for predicting the occurrence of acute Stanford type A aortic dissection(TAAD), and to construct a nomogram model for predicting the occurrence of TAAD.Methods:The clinical data of patients meeting the diagnostic criteria for TAAD admitted to Tianjin Chest Hospital from June 2016 to December 2021 and healthy people examined by the physical examination center of Tianjin Chest Hospital during the same period were retrospectively collected, and the independent risk factors for TAAD were predicted by propensity matching analysis. Univariate and multivariate Logistic regression were used to analyze the variables with statistical differences, and a nomogram model was constructed to predict the occurrence of TAAD disease according to the screened risk factors. Results:A total of 148 patients in the TAAD group and 5 690 patients in the control group were collected. After bias matching analysis, 148 pairs were successfully matched. Multivariate Logistic regression analysis was performed on the matching results. The results showed that hypertension(HBP), diabetes mellitus(T2DM), Lp(a), very low density lipoprotein(VLDL) and apolipoprotein A1/B(ApoA1/B) were independent risk factors for the development of TAAD. HBP, Lp(a) and ApoA1/B were pathogenic factors( OR 7.267, 1.010 and 2.199, P<0.05, respectively), while T2DM and VLDL were protective factors( OR 0.173 and 0.139, P<0.05). Based on the independent risk factors obtained by multi-factor Logistic regression analysis, a nomogram model of TAAD incidence was constructed. The area under ROC curve( AUC) for predicting the onset of TAAD was 81.6%(95% CI: 0.766-0.863), and the internal calibration curve was close to the standard curve. Conclusion:This model has a good degree of differentiation and calibration, which is helpful for clinicians to guide healthy people to prevent the occurrence of TAAD and provide a theoretical basis for the prevention of TAAD.

ObjectiveTo explore the possible mechanism of Tongfeng Decoction （痛风汤） for preventing and treating acute gouty arthritis. MethodsSixty Wistar male rats were divided into normal group， model group， colchicine group and low-， medium- and high-dose Tongfeng Decoction groups according to the random number table， 10 rats in each group. Tongfeng Decoction of 11.34， 22.68 and 45.36 g/kg were given by gavage to low-， medium- and high-dose Tongfeng Decoction groups， colchicine 3.15×10^-4 g/（kg·d） to the colchicine group， and normal saline 10 ml/（kg·d） to the normal group and model group respectively for 7 consecutive days. After 1 hour of gavage on day 5， rats in all groups except the normal group were modelled as acute gouty arthritis in the ankle joint of the right hind limb with the modified Coderre's method； rats in the normal group were injected with 0.2 ml of normal saline at the same location. The swelling degree of the ankle joint was measured before modelling and after 6 h， 12 h， 24 h and 48 h of modelling， respectively. HE staining was used to observe the histopathological and morphological changes in the synovial tissue of the ankle joint； immunoblotting and RT-qPCR were used to detect NOD-like receptor protein 3 （NLRP3）， apoptosis-associated speck-like protein （ASC）， cysteine aspartate protease 1 （Caspase-1）， and interleukin 1β （IL-1β） protein and mRNA expression levels in ankle synovial tissues， respectively； immunohistochemistry was used to detect the positive expression of Caspase-1 and IL-1β in ankle synovial tissues； ELISA was used to detect the tumour necrosis factor alpha （TNF-α）， IL-1β， and interleukin in serum； immunofluorescence staining was used to observe the formation of neutrophil extracellular traps （NETs） in the synovial tissues of the ankle joints. ResultsCompared with the normal group， rats in the model group showed elevated joint swelling at all time points， significantly increased inflammatory cell infiltration and the number of synovial cell layers in the synovial tissues of the ankle joints， elevated NLRP3， Caspase-1， ASC and IL-1β protein and mRNA expression， elevated positive expression of Caspase-1 and IL-1β， increased formation of NETs， and elevation of TNF-α， IL-6， and IL-1β in serum （P＜0.05）. Compared with the model group， there was an improvement in synovial cell proliferation and inflammatory cell infiltration of the ankle joint in the colchicine group， high- and medium-dose Tongfeng Decoction groups. In the colchicine group and the high-， medium- and low-dose Tongfeng Decoction groups， the degree of joint swelling， positive expression of Caspase-1， IL-1β and formation of NETs in the synovial tissue of the ankle joint， and TNF-α， IL-6 and IL-1β in serum reduced 24 h and 48 h after modelling； in colchicine group and high-dose Tongfeng Decoction group， NLRP3， ASC， Caspase-1， IL-1β protein and mRNA expression in the synovial tissues of ankle joints all reduced （P＜0.05）. The colchicine group and high-dose Tongfeng Decoction group were superior to the low-dose Tongfeng Decoction group in reducing ASC， Caspase-1， IL-1β protein and mRNA expression in the ankle synovial tissues， the positive expression of Caspase-1， as well as TNF-α， IL-6， and IL-1β level in serum （P＜0.05）. ConclusionTongfeng Decoction showed effectiveness for the prevention and treatment of acute gouty arthritis， and its mechanism may be related to the inhibition of the assembly and activation of NLRP3 inflammatory vesicles in ankle synovial tissues， the inhibition of the release of inflammatory factors， and the formation of NETs.

Objective To study the effects of Guizhi Shaoyao Zhimu Decoction on factors related to bone destruction and bone protection in rats with collagen-induced arthritis(CIA)based on osteopro-tegerin(OPG)/receptor activator of NF-κB ligand(RANKL)/receptor activator of NF-κB(RANK)signaling pathway.Methods According to the body weight,60 female Wistar rats were randomly di-vided into the following six groups:the normal group,the model group,the Triperygium wilfordii mul-tiglucoside group(0.01 g/kg),the Guizhi Shaoyao Zhimu Decoction low-dose group(8.6 g/kg),the Guizhi Shaoyao Zhimu Decoction medium-dose group(17.2 g/kg),and the Guizhi Shaoyao Zhimu Decoction high-dose group(34.4 g/kg)(n=10 rats per group).The rats in all groups except for the normal group were given 100 μg bovine type Ⅱ collagen on the 1st and 8th days to establish the CIA model,and was injected into the left foot sole and tail root of the rats.After the successful modeling,the rats were treated by gavage for 4 weeks.The general state,body weight,and arthritis index(AI)score of rats were recorded,and the contents of RANKL and OPG in rat serum were determined by enzyme-linked immunosorbent assay.The mRNA and protein expressions of RANKL,RANK,and OPG in the ankle joint were determined through real-time PCR and Western blotting,respectively.Results Com-pared with the normal group,the general state of the model group was poor,the toe swelling was obvious,the AI score was increased,the serum RANKL content was increased,the serum OPG content was de-creased,and the mRNA and protein expressions of RANKL and RANK in the ankle joint were increased(P＜0.05).Compared with the model group,the degree of toe swelling and the AI score of rats in the Guizhi Shaoyao Zhimu Decoction low-,medium-,and high-dose groups were decreased,the serum RANKL content was decreased,the serum OPG content was increased,the mRNA and protein expres-sions of RANKL and RANK in the ankle joint were decreased,the mRNA and protein expressions of OPG were increased,and the RANKL/OPG ratio of the ankle joint was decreased(P＜0.05).Conclusion Guizhi Shaoyao Zhimu Decoction can improve the destruction of joint bone in CIA rats,and its mecha-nism of action may be related to reducing RANKL level,reducing RANKL/OPG ratio,and regulating bone balance.

Objective To establish a diagnostic model for scleroderma by combining machine learning and artificial neural network based on mitochondria-related genes.Methods The GSE95065 and GSE59785 datasets of scleroderma from GEO database were used for analyzing expressions of mitochondria-related genes,and the differential genes were identified by Random forest,LASSO regression and SVM algorithms.Based on these differential genes,an artificial neural network model was constructed,and its diagnostic accuracy was evaluated by 10-fold crossover verification and ROC curve analysis using the verification dataset GSE76807.The mRNA expressions of the key genes were verified by RT-qPCR in a mouse model of scleroderma.The CIBERSORT algorithm was used to estimate the bioinformatic association between scleroderma and the screened biomarkers.Results A total of 24 differential genes were obtained,including 11 up-regulated and 13 down-regulated genes.Seven most relevant mitochondria-related genes(POLB,GSR,KRAS,NT5DC2,NOX4,IGF1,and TGM2)were screened using 3 machine learning algorithms,and the artificial neural network diagnostic model was constructed.The model showed an area under the ROC curves of 0.984 for scleroderma diagnosis(0.740 for the verification dataset and 0.980 for cross-over validation).RT-qPCR detected significant up-regulation of POLB,GSR,KRAS,NOX4,IGF1 and TGM2 mRNAs and significant down-regulation of NT5DC2 in the mouse models of scleroderma.Immune cell infiltration analysis showed that the differential genes in scleroderma were associated with follicular helper T cells,immature B cells,resting dendritic cells,memory activated CD4+T cells,M0 macrophages,monocytes,resting memory CD4+T cells and mast cell activation.Conclusion The artificial neural network diagnostic model for scleroderma established in this study provides a new perspective for exploring the pathogenesis of scleroderma.

Objective To establish a diagnostic model for scleroderma by combining machine learning and artificial neural network based on mitochondria-related genes.Methods The GSE95065 and GSE59785 datasets of scleroderma from GEO database were used for analyzing expressions of mitochondria-related genes,and the differential genes were identified by Random forest,LASSO regression and SVM algorithms.Based on these differential genes,an artificial neural network model was constructed,and its diagnostic accuracy was evaluated by 10-fold crossover verification and ROC curve analysis using the verification dataset GSE76807.The mRNA expressions of the key genes were verified by RT-qPCR in a mouse model of scleroderma.The CIBERSORT algorithm was used to estimate the bioinformatic association between scleroderma and the screened biomarkers.Results A total of 24 differential genes were obtained,including 11 up-regulated and 13 down-regulated genes.Seven most relevant mitochondria-related genes(POLB,GSR,KRAS,NT5DC2,NOX4,IGF1,and TGM2)were screened using 3 machine learning algorithms,and the artificial neural network diagnostic model was constructed.The model showed an area under the ROC curves of 0.984 for scleroderma diagnosis(0.740 for the verification dataset and 0.980 for cross-over validation).RT-qPCR detected significant up-regulation of POLB,GSR,KRAS,NOX4,IGF1 and TGM2 mRNAs and significant down-regulation of NT5DC2 in the mouse models of scleroderma.Immune cell infiltration analysis showed that the differential genes in scleroderma were associated with follicular helper T cells,immature B cells,resting dendritic cells,memory activated CD4+T cells,M0 macrophages,monocytes,resting memory CD4+T cells and mast cell activation.Conclusion The artificial neural network diagnostic model for scleroderma established in this study provides a new perspective for exploring the pathogenesis of scleroderma.

Objective:The transjugular or transfemoral approach is used as a common method for hepatic venous pressure gradient (HVPG) measurement in current practice. This study aims to confirm the safety and effectiveness of measuring HVPG via the forearm venous approach.Methods:Prospective recruitment was conducted for patients with cirrhosis who underwent HVPG measurement via the forearm venous approach at six hospitals in China and Japan from September 2020 to December 2020. Patients' clinical baseline information and HVPG measurement data were collected. The right median cubital vein or basilic vein approach for all enrolled patients was selected. The HVPG standard process was used to measure pressure. Research data were analyzed using SPSS 22.0 statistical software. Quantitative data were used to represent medians (interquartile ranges), while qualitative data were used to represent frequency and rates. The correlation between two sets of data was analyzed using Pearson correlation analysis.Results:A total of 43 cases were enrolled in this study. Of these, 41 (95.3%) successfully underwent HVPG measurement via the forearm venous approach. None of the patients had any serious complications. The median operation time for HVPG detection via forearm vein was 18.0 minutes (12.3~38.8 minutes). This study confirmed that HVPG was positively closely related to Child-Pugh score ( r = 0.47, P = 0.002), albumin-bilirubin score ( r = 0.37, P = 0.001), Lok index ( r = 0.36, P = 0.02), liver stiffness ( r = 0.58, P = 0.01), and spleen stiffness ( r = 0.77, P = 0.01), while negatively correlated with albumin ( r = -0.42, P = 0.006). Conclusion:The results of this multi-centre retrospective study suggest that HVPG measurement via the forearm venous approach is safe and feasible.

OBJECTIVE To investigate the effects of β-sitosterol on the function of rheumatoid arthritis （RA） fibroblastic synoviocytes MH7A cells and its mechanism. METHODS Network pharmacology was adopted to screen the targets of β-sitosterol and the targets for the treatment of RA. After the intersection of them， topological analysis was performed to find the most critical target in the treatment of RA. MH7A cells were treated with different concentrations （0， 5， 10， 20， 40 μmol/L） of β-sitosterol， and CCK-8 was used to assay cell viability for screening the optimal concentration of β-sitosterol. MH7A cells were induced by 10 ng/mL TNF-α in vitro and treated with β-sitosterol （the optimum concentration）. CCK-8 and EdU were used to detect the ability of cell proliferation. Scratch experiment and Transwell invasion assay were used to analyze cell migration and invasion. The levels of interleukin-1β （IL-1β） and IL-6 in cell supernatant were detected by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expressions of peroxisome proliferator-activated receptor α （PPARα） were measured by qRT-PCR and Western blot， respectively. The siRNA targeting PPARα was transfected into MH7A cells， and the effects of β-sitosterol on cell proliferation， migration， invasion， the secretion of inflammatory factors and the expression of PPARα after PPARα knockdown were detected by the above experimental methods. RESULTS PPARα was the most critical target of β-sitosterol in the treatment of RA. The optimal concentration of β-sitosterol was 20 μmol/L. Compared with model group， β-sitosterol decreased the viability of MH7A cells， and the number of proliferating cells also decreased significantly （P＜0.05）； the cell migration rate and the number of cell invasion decreased significantly （P＜0.05）. The levels of IL-1β and IL-6 were also significantly decreased （P＜0.05）， and the mRNA 15 and protein expression levels of PPARα were significantly increased （P＜0.05）. Compared with negative control small interfering RNA group， after PPARα knockdown， the cell viability increased by about 35.6% （P＜0.05）， the number of cell proliferation， the cell migration rate and the number of cell invasion increased significantly （P＜0.05）， and the levels of IL-1β and IL-6 also increased significantly （P＜0.05）. CONCLUSIONS β-sitosterol could effectively inhibit the proliferation， migration， invasion and secretion of inflammatory factors in MH7A cells， the mechanism of which may be associated with activating PPARα pathway.

AIM: To investigate the clinical features of dry eye in patients with type 2 diabetes mellitus complicated with peripheral neuropathy.METHOD: Prospective cohort study. A total of 192 patients with type 2 diabetes were enrolled in the Department of Endocrinology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine from July 2021 to March 2022. The right eyes of all patients were selected as the observation eye, among which 122 patients were diagnosed with diabetic peripheral neuropathy(DPN)and 70 patients were diagnosed with non-diabetic peripheral neuropathy(NDPN). The score of ocular surface disease index(OSDI), tear meniscus height, tear meniscus width, corneal epithelial thickness, corneal endothelial cell density, tear secretion test(Schirmer Ⅰ test, SⅠt), corneal sensitivity, meibomian gland function status score, tear film breakup time(BUT), corneal fluorescein sodium staining score and Toronto clinical scoring system(TCSS)score were compared between two groups. The correlation between OSDI score and TCSS score in type 2 diabetes patients was analyzed as well.RESULTS: The morbidity of dry eye in the DPN group(55 eyes, 45.1%)was significantly higher than that of NDPN group(20 eyes, 28.6%; χ2=5.094, P=0.024), BUT and corneal sensitivity score of DPN were lower than NDPN group(P<0.001), meanwhile, corneal staining score and meibomian gland function score were higher than NDPN group(P<0.001). OSDI scores of all subjects were negatively correlated with TCSS scores(rs=-0.233, P=0.002), and OSDI scores of DPN group were negatively correlated with TCSS scores(rs=-0.511, P<0.001), but there was no significant correlation between the two scores of NDPN patients(rs=0.007, P=0.957).CONCLUSIONS: DPN patients are more likely to develop dry eye than NDPN patients. OSDI score is not an accurate evaluation index for type 2 diabetes patients, especially for DPN patients.

Objective:To study the impact and the mechanism of splenectomy combined with pericardial devascularization on cirrhotic livers.Methods:Serum samples and clinical data were collected preoperatively and postoperatively from 54 patients with cirrhosis who underwent splenectomy combined with pericardial devascularization from May 2013 to Oct 2014 at Beijing You’an Hospital, Capital Medical University. Changes in hepatic arterial and portal venous blood flow, liver function and fibroscan results were analyzed. The levels of nitric oxide (NO), endothelin-1 (ET-1), interleukin-6 (IL-6), hepatocyte growth factor (HGF), transforming growth factor-β1 (TGF-β1) and matrix metalloproteinase 1 (MMP1) were measured.Results:There were 31 males and 23 females, aged(45.48±10.21)years. Free portal vein pressure decreased significantly from (37.0±7.1) cmH 2O (1 cmH 2O=0.098 kPa) to (26.1±5.7) cmH 2O after surgery ( P<0.05). Significant increases in postoperative lumen diameter (4.0±1.0) mm vs (3.1±0.7) mm were observed, accompanied by increase in peak flow velocity and blood flow of the hepatic artery. Significant deductions in lumen diameter (11.9±2.0) mm vs (13.1±1.9) mm, accompanied by reduction of peak flow velocity and blood flow of the portal vein were observed following surgery (all P<0.05). The NO level was significantly elevated immediately after splenectomy and was subsequently remained at high levels. The ET-1 level decreased 2 days after surgery and became fluctuated at low levels. The IL-6 and HGF levels increased significantly 2 days after surgery and decreased gradually after 7 days and 1 month, respectively. The TGF-β1 and the MMP1 levels increased after surgery. The endotoxin level decreased significantly after surgery (all P<0.05). Conclusion:Splenectomy combined with pericardial devascularization induced hepatic blood flow restoration, hepatocyte regeneration and reversal of fibrosis in cirrhotic livers. Splenectomy has a protective effect on cirrhotic liver when combined with pericardial devascularization.