Aim To elucidate the biological effects of insulin-like growth factor-1 ( IGF-1 ) and basic fibro-blast growth factor (bFGF) alone or in combination on the differentiation of bone mesenchymal stem cells (BMSCs) into cardiomyocytes (CMs), and to explore the mechanism in the differentiation process of BMSCs into CMs induced by IGF-1 or bFGF. Methods After four weeks of BMSCs induced by induction differentiation medium ( with or without LY294002) containing IGF-1 and/or bFGF, the expression levels of proteins associated with the cardiomyogenic phenotype in BMSCs were detected via immunocytochemistry, immuno-fluorescence staining, and Western blot. Meanwhile, the expression levels of pathway related proteins were detected by Western blot. The cell ultrastructure was observed by transmission electron microscopy (TEM) . The expression levels of myocardial specific genes were measured via RT-qPCR. Results Compared with the control group, the expression levels of myocardial specific proteins and genes significantly increased in the IGF-1, bFGF and combination groups and were the highest in the coinduction group. The TEM of the conduction group showed parallel myofilaments, mitochondria, endoplasmic reticulum and so on. The additon of LY294002 decreased the expression levels of myocardial specific proteins, genes and phosphorylation Akt. Conclusions The effect of IGF-1 combined with bFGF on the differentiation of BMSCs into CMs is markedly better than that induced by IGF-1 or bFGF a-lone, and the differentiation process may depend on the PI3K/Akt signaling pathway.

Galectin-3 (Gal-3) belongs to the galectin family and is specific in binding β-galactoside. Through its C-terminal domain, Gal-3 binds to the galactoside group of the glycosylated insulin receptor (IR) and inhibits IR signaling pathway, which leads to the insulin resistance. Thus, Gal-3 is a potential therapeutic target for the treatment of insulin resistance and type 2 diabetes. Here we report a simple Gal-3 screening model based on the property that Gal-3 binds to the galactoside. We expressed and purified human Gal-3 in Escherichia coli (E.coli), and labeled it with fluorescein isothiocyanate (FITC) in vitro. After incubating FITC labeled Gal-3 (Gal-3-FITC) with PANC-1 cells, which express glycosylated membrane protein, PANC-1 cells started to show green fluorescent signal due to the Gal-3-FITC binding to the glycosylated membrane protein. Gal-3 inhibitor disrupts the binding of Gal-3-FITC and PANC1 cells, subsequently leads to the decrease of the fluorescent signal in PANC-1 cells. We can evaluate the inhibitory efficiency of Gal-3 inhibitors through measurement of the fluorescent signal. Further studies show this model is simple, stable, and repeatable with a Z' factor between 0.7 and 0.85. In sum, we have successfully established an in vitro high-throughput screening model for Gal-3 inhibitors.

Objective:To observe the clinical characteristics and guideline compliance of chronic obstructive pulmonary disease (COPD) patients with initial triple therapy in real-life world.Methods:This study is a cross-sectional study. The subjects of the study were COPD patients admitted to 13 hospitals in Hunan Province and Guangxi Zhuang Autonomous Region from December 2016 to December 2021. The initial treatment was triple inhaled drugs. The data collected included gender, age, diagnosis, body mass index (BMI), history of acute exacerbation (AE) in the past year, pulmonary function, COPD Assessment Test (CAT) score, modified British Medical Research Council Dyspnea Questionnaire (mMRC), inhaled drugs and other indicators. The characteristics and differences of COPD patients before and after 2020 were analyzed.Results:7 184 patients with COPD were enrolled in this study, including 2 409 COPD patients treated with initial triple therapy, accounting for 33.5%(2 409/7 184). Taking January 1st, 2020 as the cut-off point, 1 825 COPD patients (75.8%) received initial treatment with triple inhaled drugs before 2020 and 584 patients (24.2%) after 2020 were included in this study. Compared with COPD patients before 2020, the COPD patients after 2020 had higher FEV 1% [(40.9±15.5 )% vs (39.3±15.5)%, P=0.040], lower CAT [(15.8±6.5)point vs (17.5±6.2)point, P<0.001], less AE in the past year [1(0, 2)times vs 1(0, 2)times, P=0.001] and higher rate of non-AE [255(43.7%) vs 581(37.1%), P=0.006]. In addition, before 2020, patients with COPD were mainly treated with open triple drugs (1 825/1 825, 100%); after 2020, 306 patients (52.4%) received open triple inhaled drugs, and 278 patients (47.6%) received closed triple inhaled drugs. Conclusions:In real-life world, most of patients with COPD treated with triple therapy have severe lung function, obvious symptoms and high risk of acute exacerbation. The real-world prescribing of triple therapy in patients with COPD does not always reflect recommendations in guidelines and strategies, and overtreatment is common. After 2020, prescribing triple therapy for COPD patients is more positive and worse consistency with guideline.

OBJECTIVES: To investigate the incidence of extrauterine growth retardation (EUGR) and its risk factors in very preterm infants (VPIs) during hospitalization in China. METHODS: A prospective multicenter study was performed on the medical data of 2 514 VPIs who were hospitalized in the department of neonatology in 28 hospitals from 7 areas of China between September 2019 and December 2020. According to the presence or absence of EUGR based on the evaluation of body weight at the corrected gestational age of 36 weeks or at discharge, the VPIs were classified to two groups: EUGR group (n=1 189) and non-EUGR (n=1 325). The clinical features were compared between the two groups, and the incidence of EUGR and risk factors for EUGR were examined. RESULTS: The incidence of EUGR was 47.30% (1 189/2 514) evaluated by weight. The multivariate logistic regression analysis showed that higher weight growth velocity after regaining birth weight and higher cumulative calorie intake during the first week of hospitalization were protective factors against EUGR (P<0.05), while small-for-gestational-age birth, prolonged time to the initiation of total enteral feeding, prolonged cumulative fasting time, lower breast milk intake before starting human milk fortifiers, prolonged time to the initiation of full fortified feeding, and moderate-to-severe bronchopulmonary dysplasia were risk factors for EUGR (P<0.05). CONCLUSIONS It is crucial to reduce the incidence of EUGR by achieving total enteral feeding as early as possible, strengthening breastfeeding, increasing calorie intake in the first week after birth, improving the velocity of weight gain, and preventing moderate-severe bronchopulmonary dysplasia in VPIs.
Female ; Fetal Growth Retardation ; Gestational Age ; Hospitalization ; Humans ; Incidence ; Infant ; Infant, Newborn ; Infant, Premature ; Infant, Very Low Birth Weight ; Prospective Studies ; Risk Factors

The GPCR family component leukotriene B4 receptor 1 (LTB4R1) is the receptor of leukotriene B4 (LTB4), the metabolic product of ω6 fatty acid. LTB4R1 is a potential therapeutic target for the treatment of insulin resistance, chronic inflammation and type 2 diabetes. Here we established a LTB4R1 inhibitor screen model based on the GPCR family protein property that its activation causes the cytosolic escalation of calcium. The cytosolic calcium probe Fluo-8 represents the change of calcium ion. After adding LTB4, the fluorescent signal of Fluo-8 in the CHO cells which are co-transfected with LTB4R1 and Gα16 will change with the increase of cytosolic calcium, and LTB4R1 inhibitor blocked the effect of LTB4 on fluorescent signal of Fluo-8 in the CHO cells. Here, we used 0.2% DMSO as a negative control, and cp-105696 as a positive control in the screen model. After stimulation with LTB4, the Fluo-8 signal in 0.2% DMSO treated CHO cells increased 2 fold and fell back slowly, while the signal in inhibitor (cp-105696) treated cells was not induced by LTB4. The results showed that LTB4 increased the cytosolic calcium detected by Fluo-8 in a dose dependent manner. Similarly, cp-105696 inhibited the Fluo-8 signal dose dependently, indicating that this method can quantify the inhibitory activity of the compounds. The Z'-factor, reflecting the robustness of the screen model, was 0.777 with a series of experiments. In sum, we over-expressed LTB4R1α and Gα16 in CHO cell, used Fluo-8 to detect the calcium signal activated by LTB4, and established the in vitro screen model for LTB4 receptor 1.

Objective:To analyze the risk factors of bronchopulmonary dysplasia(BPD)in very preterm infants(VPI), and to provide scientific basis for the prevention and treatment of BPD in VPI.Methods:A prospective multicenter study was designed to collect the clinical data of VPI in department of neonatology of 28 hospitals in 7 regions from September 2019 to December 2020.According to the continuous oxygen dependence at 28 days after birth, VPI were divided into non BPD group and BPD group, and the risk factors of BPD in VPI were analyzed.Results:A total of 2 514 cases of VPI including 1 364 cases without BPD and 1 150 cases with BPD were enrolled.The incidence of BPD was 45.7%.The smaller the gestational age and weight, the higher the incidence of BPD( P<0.001). Compared with non BPD group, the average birth age, weight and cesarean section rate in BPD group were lower, and the incidence of male infants, small for gestational age and 5-minute apgar score≤7 were higher( P<0.01). In BPD group, the incidences of neonatal respiratory distress syndrome(NRDS), hemodynamically significant patent ductus arteriosus, retinopathy of prematurity, feeding intolerance, extrauterine growth restriction, grade Ⅲ~Ⅳ intracranial hemorrhage, anemia, early-onset and late-onset sepsis, nosocomial infection, parenteral nutrition-associated cholestasis were higher( P<0.05), the use of pulmonary surfactant(PS), postnatal hormone exposure, anemia and blood transfusion were also higher, and the time of invasive and non-invasive mechanical ventilation, oxygen use and total hospital stay were longer( P<0.001). The time of starting enteral nutrition, cumulative fasting days, days of reaching total enteral nutrition, days of continuous parenteral nutrition, days of reaching 110 kcal/(kg·d) total calorie, days of reaching 110 kcal/(kg·d) oral calorie were longer and the breastfeeding rate was lower in BPD group than those in non BPD group( P<0.001). The cumulative doses of amino acid and fat emulsion during the first week of hospitalization were higher in BPD group( P<0.001). Multivariate Logistic regression analysis showed that NRDS, invasive mechanical ventilation, age of reaching total enteral nutrition, anemia and blood transfusion were the independent risk factors for BPD in VPI, and older gestational age was the protective factor for BPD. Conclusion:Strengthening perinatal management, avoiding premature delivery and severe NRDS, shortening the time of invasive mechanical ventilation, paying attention to enteral nutrition management, reaching whole intestinal feeding as soon as possible, and strictly mastering the indications of blood transfusion are very important to reduce the incidence of BPD in VPI.

Objective: To explore the mechanism of herbal cake-partitioned moxibustion in Crohn disease (CD) treatment by observing the effect of herbal cake-partitioned moxibustion on protein expressions of colonic M2 macrophage marker CD206, AMP-activated protein kinase (AMPK) and tuberous sclerosis complex (TSC) 2. Methods: Twenty-six specific pathogen free male rats were randomly divided into a normal group, a model group and a herbal cake-partitioned moxibustion group. The CD model was prepared by enema with the mixture of 5％ (W/V) 2,4,6- trinitrobenzene sulfonic acid (TNBS) and 50％ ethanol at 2:1 (volume ratio). After the model was successfully prepared, rats in the herbal cake-partitioned moxibustion group received herbal cake-partitioned moxibustion at Qihai (CV 6) and bilateral Tianshu (ST 25). Hematoxylin-eosin (HE) staining was used to observe the histopathological changes of rat colon; immunohistochemical technique was used to detect the expression of colonic CD206 protein; Western blot, immunofluorescence, and real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) technologies were used to detect the protein and mRNA expressions of colonic AMPK and TSC2. Results: Compared with the normal group, rats in the model group showed damaged colonic mucosa, missing of the epithelial layer, thickened submucosa, vascular proliferation, massive infiltration of monocytes and lymphocytes, and cracked ulcers that reached the muscle layer. Rats in the herbal cake-partitioned moxibustion group showed reduced intestinal inflammation and healing intestinal epithelium ulcers. Compared with the normal group, rat colonic CD206 protein expression, and the protein and mRNA expressions of colonic AMPK and TSC2 were decreased in the model group (all P<0.01); compared with the model group, rat colonic CD206 protein expression was increased (P<0.01), as well as the protein and mRNA expressions of AMPK and TSC2 in the herbal cake-partitioned moxibustion (all P<0.05). Conclusion: Herbal cake-partitioned moxibustion can reduce intestinal inflammation in CD rats, increase colonic CD206 protein expression, and up-regulate the protein and mRNA expressions of colonic AMPK and TSC2.

OBJECTIVE: To evaluate the effect of comprehensive therapy based on Chinese medicine (CM) patterns on self-efficacy and satisfaction with its effectiveness in patients with chronic obstructive pulmonary disease (COPD). METHODS: A total of 216 patients were randomly divided into the trial group (n =108) and the control group (n=108) based on the stratified and block randomization design. Patients in the trial group were treated with conventional Western medicine combined with Bufei Jianpi Granules (), Bufei Yishen Granules (), and Yiqi Zishen Granules () according to the CM patterns respectively, and patients in the control group were treated with conventional Western medicine. The COPD Self-Efficacy Scale (CSES) and the Effectiveness Satisfaction Questionnaire for COPD (ESQ-COPD) were employed in a 6-month treatment and in further 6 month follow-up visit. RESULTS: Among the 216 patients, 191 patients (97 in the trial group and 94 in the control group) fully completed the study. After 12-month treatment and follow-up, the mean scores of the trial group all continued to increase over time, which were significantly higher than those of the control group (P <0.05), and the improvement in the following trial group domain: negative affect domain (12.13%), intense emotional arousal domain (12.21%), physical exertion domain (11.72%), weather/environmental domain (13.77%), behavioral risk domain (7.67%) and total score (10.65%). The trial group also exhibited significantly higher mean scores in the ESQ-COPD (P <0.05) and the improvement in the following domain: capacity for life and work domain (30.59%), clinical symptoms domain (53.52%), effect of therapy domain (35.95%), convenience of therapy domain (35.54%), and whole effect domain (52.47%). CONCLUSIONS Bufei Jianpi Granules, Bufei Yishen Granules and Yiqi Zishen Granules can improve the self-efficacy and satisfaction of COPD patients.

OBJECTIVE: To construct a W203X-mutant mouse model of cblC type methylmalonic acidemia based on the CRISPR/Cas9 technology. METHODS: At first, BLAST was used to compare the conservative nature of the cblC gene and protein sequences in humans and mice, and then, the CRISPR/Cas9 technology was used for microinjection of mouse fertilized eggs to obtain heterozygous F1 mice. Hybridization was performed for these mice to obtain homozygous W203X-mutant mice. The blood level of the metabolite propionyl carnitine (C3) was measured for homozygous mutant mice, heterozygous littermates, and wild-type mice. RESULTS: The gene and protein sequences of MMACHC, the pathogenic gene for cblC type methylmalonic acidemia, were highly conserved in humans and mice. The homozygous W203X-mutant mice were successfully obtained by the CRISPR/Cas9 technology, and there was a significant increase in C3 in these mice at 24 hours after birth (P<0.001). CONCLUSIONS A W203X-mutant mouse model of cblC type methylmalonic acidemia is successfully constructed by the CRISPR/Cas9 technology.
Amino Acid Metabolism, Inborn Errors ; Animals ; CRISPR-Cas Systems ; Carrier Proteins ; Heterozygote ; Mice ; Mutation

Objective To investigate the role of microRNA-34a (miR-34a) in the proliferation of vascular smooth muscle cells (VSMCs) induced by low shear stress (LowSS). Methods Using co-culture parallel plate flow chamber system, endothelial cells (ECs) and VSMCs were co-cultured and applied with normal shear stress (1.5 Pa) and LowSS (0.5 Pa) for 12 h. The expression of proliferating cell nuclear antigen (PCNA) in the co-cultured VSMCs was detected by Western blotting to determine the proliferation capacity of VSMCs. Real-time PCR was used to examine the miR levels of miR-34a in the co-cultured VSMCs. The target proteins of miR-34a were predicted by TargetScan, miRWalk and some other websites. Western blotting was used to detect expression of Forkhead box j2 (Foxj2) in the co-cultured VSMCs. Mimics and inhibitor were used to up-regulate or inhibit the expression of miR-34a, and then the expression of Foxj2 and PCNA was detected by Western blotting to verify the regulation relationship between miR 34a and Foxj2. Results Compared with NSS, LowSS promoted the PCNA expression and significantly up-regulated the miR-34a expression in the co-cultured VSMCs. Foxj2 was predicted to be the downstream target protein of miR-34a by TargetScan, miRWalk and some other websites. Foxj2 expression decreased significantly in the co-cultured VSMCs under LowSS application. Under static condition, the expression of Foxj2 obviously decreased and the expression of PCNA obviously increased by up-regulating miR-34a expression in VSMCs. While inhibiting the expression of miR-34a in VSMCs would result in a significant increase in the expression of Foxj2 and a significant decrease in the expression of PCNA. Conclusions LowSS can promote the proliferation of VSMCs by regulating miR-34a and target protein Foxj2 in the co-cultured VSMCs. This research finding will provide new mechanobiological experimental reference for further illustrating the pathogenesis of atherosclerosis and finding the therapeutic targets for drugs.