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Objective To investigate the intervention effect of Xuanfuhua decoction on mice with nonalcoholic steatohepatitis (NASH) induced by high-fat, high-fructose, and high-cholesterol diet. Methods A total of 32 male C57/BL6J mice were randomly divided into normal group, model group, Xuanfuhua decoction group, and obeticholic acid group, with 8 mice in each group. Since week 24 of modeling using high-fat, high-fructose, and high-cholesterol diet, each group was given the corresponding drug for intervention at a dose of 14.19 g/kg by gavage for the Xuanfuhua decoction group and 10 mg/kg by gavage for the obeticholic acid group and a volume of 20 mL/kg for gavage, once a day for 6 consecutive weeks. HE staining, oil red O staining, Sirius Red staining, and Masson staining were used to observe the pathological changes, lipid deposition, and collagen deposition of liver tissue; related kits were used to measure the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and glucose, as well as the content of TG and hydroxyproline (Hyp) in liver tissue; quantitative real-time PCR was used to measure the expression of genes associated with lipid metabolism, inflammation, and fibrosis in liver tissue; immunohistochemical staining was used to observe the positive expression of F4/80 and α-SMA in liver tissue. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t -test was used for further comparison between two groups. Results Compared with the normal group, the model group had significant increases in body weight, liver wet weight, and serum levels of AST, ALT, TC, TG, LDL-C and glucose (all P < 0.01). HE staining showed hepatocyte steatosis, a large number of fat vacuoles, hepatocyte ballooning degeneration, and inflammatory cell infiltration in liver tissue of the mice in the model group, and the model group had a significant increase in NAFLD activity score (NAS) compared with the normal group ( P < 0.01). Oil red O staining showed the deposition of a large number of red lipid droplets with different sizes in hepatocytes of the mice in the model group, and compared with the normal group, the model group had significant increases in the area percentage of oil red O staining and the content of TG in the liver ( P < 0.01). Sirius Red staining and Masson staining showed significant collagen fiber hyperplasia in the perisinusoidal area, the central vein, and the portal area in the model group, and the model group had a significant increase in the content of Hyp in liver tissue compared with the normal group ( P < 0.05). Compared with the model group, the Xuanfuhua decoction group had significant reductions in the serum levels of AST, ALT, TC, TG, LDL-C, and glucose (all P < 0.05), significant improvements in hepatic steatosis, inflammatory infiltration, lipid droplet deposition, and collagen fiber hyperplasia, and significant reductions in NAS score, area percentage of oil red O staining, and content of TG and Hyp in the liver (all P < 0.05). Compared with the normal group, the model group had significant increases in the mRNA expression levels of lipid metabolism-related genes (SREBP-1c, FASN, SCD-1, PPAR-γ, and CD36), inflammation-related genes (F4/80, TNF-α, CCL2, and CD11b), and the fibrosis-related gene α-SMA (all P < 0.05), and immunohistochemical staining showed significant increases in the positive expression of F4/80 and α-SMA ( P < 0.01). Compared with the model group, the Xuanfuhua decoction group had significant reductions in the mRNA expression levels of SREBP-1c, FASN, SCD-1, PPAR-γ, CD36, F4/80, TNF-α, CCL2, CD11b, and α-SMA in liver tissue (all P < 0.05), and immunohistochemical staining showed significant reductions in the positive expression of F4/80 and α-SMA ( P < 0.01). Conclusion Xuanfuhua decoction has a good intervention effect on mice with NASH induced by high fat, high fructose, and high-cholesterol diet and can significantly inhibit hepatic lipid deposition, inflammatory response, and liver fibrosis.
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Objective:To investigate the effect of lncRNA HOTTIP on the radiosensitivity of four non-small cell lung cancer cell lines cultured in vitro by regulating the expression of miR-663a. Methods:Four non-small cell lung cancer cell lines (H838, H157, A549, and H1299) were irradiated with different radiation intensities (0, 2, 4, 6, and 8 Gy). Cell survival was detected by colony formation assay. The expression levels of HOTTIP and miR-663a were detected by qRT-PCR. A549 and H1299 cells were selected for the subsequent experiment. After silencing HOTTIP and/or over-expressing miR-663a, cell survival was detected by colony formation assay. Cell apoptosis was detected by flow cytometry. The expression levels of Cleaved caspase-3, Cleaved PARP andγ-H 2AX were quantitatively measured by Western blot. The targeting relationship between HOTTIP and miR-663a was vefiried by dual luciferase reporter assay and qRT-PCR. Results:The expression of HOTTIP was up-regulated, whereas that of miR-663a was down-regulated in the radiation-resistant H157, A549 and H1299 cells. Silencing HOTTIP or over-expressing miR-663a inhibited the survival of A549 and H1299 cells (radiosensitization ratios were 1.562 and 1.507, respectively), promoted the expression of Cleaved caspase-3, Cleaved PARP and γ-H 2AX, and accelerated cell apoptosis induced by radiation exposure. miR-663a was a target gene of HOTTIP, and HOTTIP negatively regulated the expression of miR-663a. The inhibition of miR-663a reversed the effect of silencing HOTTIP on the radiosensitivity of non-small cell lung cancer cells. Conclusion:Silencing HOTTIP can suppress the survival of non-small cell lung cancer cells and promotes cell apoptosis by up-regulating the expression of miR-663a, thereby enhancing the radiosensitivity of non-small cell lung cancer cell lines.
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Objectiv e:To investigate the effect of different culture conditions on the differentiation of Treg and Th17 to lay a foundation for exploring the methods to reverse the immune tolerance induced by tumor microenvironment.Methods:The IL-6 gene was cloned and stablely transferred into the tumor cell line expressing TGF-β.The conditioned mediums ( CM) were prepared by collecting the culture supernatants of tumor cell lines with or without IL-6 expression and used in the in vitro culture of peripheral blood mononuclear cells ( PBMC ) .The changes of Treg and Th17 in PBMC treated with different CM were detected with flow cytometry ( FCM) .Results:The expression of TGF-βin BEL-7402 was higher than that in HepG2.Thus the BEL-7402 was selected for preparation of cell line stablely transfected with IL -6 gene.ELISA detection confirmed the effective expression of IL -6 by the identified cell lines.It was showed that the Treg increased in PBMC treated with culture supernatants of tumor cells .However,the presence of IL-6 reversed the increase of Treg and promoted the differentiation of Th 17.Conclusion: The culture supernatants of tumor cells increases the proportion of Treg.However,the presence of IL-6 in this CM can reverse the increase of Treg and raise the proportion of Th 17.