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Objective:To investigate the possible role of long non-coding RNA (LncRNA) 00602 in promoting browning in adipocytes induced by adenovirus type 36 (Ad36).Methods:According to Ad36 infection, adipose tissue samples of obese patients were divided into Ad36-negative group and Ad36-infected group. Realtime fluorescent quantitative PCR (qRT-PCR) was used to detect the changes in the expression of LncRNA00602 mRNA in omental adipose tissue of the two groups, and analyze the differences between the two groups. The correlation between waist-to-hip ratio, systolic blood pressure, diastolic blood pressure, fasting blood glucose, triacylglyceride and other indicators of the patients in the group with LncRNA00602 mRNA expression were analyzed. HE staining was used to detect the size of adipocytes in the omental adipose tissue of the Ad36 negative group and the Ad36 infection group. qRT-PCR and Western blotting were used to detect the mRNA and protein expression levels of uncoupling protein 1 (UCP1) and PR domain containing 16 (PRDM16) in omental adipose tissue of two groups of patients. Human adipose-derived stem cells (hADSC) were isolated and cultured, using Ad36 to induce differentiation, and divided into control group and LncRNA00602 knockdown group. On 0, 2, and 4 days after LncRNA00602 knockdown, fluoroboron dipyrrole (BODIPY) and mitochondrial red fluorescence (Mito-Tracker Red) were used to stain intracellular lipid droplets and mitochondria. At the same time, qRT-PCR and Western blotting were used to detect changes in the expression of UCP1 and PRDM16.Results:The expression of LncRNA00602 gene in the Ad36 infection group was higher than that in the Ad36 negative group (all P<0.05). The expression of LncRNA00602 in the Ad36 negative group was not significantly different from the above clinical indicators, while the expression of LncRNA00602 was negatively correlated with serum fasting blood glucose and triacylglyceride ( r=-0.522, -0.486, P<0.05) in the Ad36 infection group; HE staining showed that the average adipocyte area of the Ad36 infection group was smaller than that of the Ad36 negative group. At the same time, UCP1 and PRDM16 gene expression were higher than the negative group (all P<0.05). At the cellular level, on the 2nd and 4th days after knockdown of LncRNA00602, the lipid droplet area of adipocytes in the LncRNA00602 knockdown group was larger than that of the control group, the number of mitochondria decreased compared with the control group, and difference was statistically significant ( P<0.05 or P<0.01); Compared with the control group, there was significantly lower expression of the browning marker genes UCP1, PRDM16, and protein in the adipocytes in the LncRNA00602 knockdown group (all P<0.05). Conclusion:In Ad36-induced adipocyte differentiation, LncRNA00602 may positively regulate the expression of UCP1, PRDM16 and lipid droplet metabolism, and promote the browning of adipocytes.
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Objective The aim of this study is to explore the hypoglycemic effect of active components of Anoectochilus roxburghii on zebrafish models. Methods Anoectochilus roxburghii components were extracted and separated into three groups: the alcohol extraction group, macromolecular polysaccharide group (≥ 5 ×103) and small molecular polysaccharide group (＜5×103). Zebrafish embryos were exposed to 2% glucose solution (2% Glu) at 24 h to imitate acute hyperglycemia phenotype, and then treated with the three Anoectochilus roxburghii components. Based on this high-glucose model, the zebrafish embryos at 72 h were collected to detect the whole tissue glucose value. Furthermore, semi-quantitative PCR and whole mount in situ hybridization were performed to detect the expression of mRNA levels of glycometabolism-related genes. Results An acute diabetic zebrafish model was induced by high glucose stress. In this model, some key factors during glycometabolism such as insulin, pck-1 and pdx-1 were significantly affected, while the alcohol extracts of Anoectochilus roxburghii obviously reversed these abnormalities induced by high glucose stress, even to normal levels. Conclusions The alcohol extracts of Anoectochilus roxburghii has obvious hypoglycemic effect on diabetic zebrafish model. Our result suggest that Anoectochilus roxburghii has a potential application in the hypoglycemic drug screening.
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AIM: To study the influence of injection agent of pidotimod on function of the immune-system in normal and immune-depressed mice, and to investigate the mechanisms of the immunomodulating action of pidotimod. METHODS: The expressions of TNF-? and IL-6 in mice spleen was detected by RT-PCR method. The effects of pidotimod on phagocytosis functions of mice peritoneal exudate cells was investigated by neutral red phagocytosis. The lymphocyte proliferations induced by Con A or LPS were detected by MTT method. Adopt the serum haemolysis to measure the production of serum antibody. RESULTS: The normal and immune-depressed mice were treated for 14 days with different dosages of pidotimod by injection. Pidotimod can significantly increase the expressions of TNF-? and IL-6 of spleen, potentiate phagocytosis of the peritoneal exudate cells, potentiate the lymphocyte proliferations ability induced by Con A or LPS. CONCLUSION: Pidotimod can potentiate amelioration normal and immune-depressed mice immunesystem function and increase expressions of TNF-? and IL-6 of spleen.
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AIM: To develop a reliable primary cultured hepatocytes model in vitro for liver metastasis research. METHODS: Hepatocytes were isolated by a modification of the two-step collagenase perfusion method. The apoptosis and cell cycle of hepatocytes were measured with flow cytometry. The proliferation of hepatocytes was detected by SRB method. RESULTS: The viability and purity of hepatocytes were 90% and 95%,respectively. The result of flow cytometry analysis showed that there was little apoptosis in hepatocytes and most of hepatocytes were in G_0/G_1 phase. The proliferation and albumin-secreting function of hepatocyte cultured by low glucose DMEM and high glucose DMEM were higher than that of cultured by RPMI1640 during 1 to 6 day, but there was no significant different between low glucose DMEM group and high glucose DMEM group. CONCLUSION: Hepatocytes have higher purity and viability with the normal biological activity for about 6 days by this method and it may be a cell model for the study of liver metastasis in vitro.