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ObjectiveTo study the effect of Qizhu Kang'ai prescription （QZAP） on the gluconeogenesis enzyme phosphoenolpyruvate carboxykinase 1 （PCK1） in the liver of mouse model of liver cancer induced by diethylnitrosamine （DEN） combined with carbon tetrachloride （CCl4） and Huh7 cells of human liver cancer， so as to explore the mechanism on regulating metabolic reprogramming and inhibiting cell proliferation of liver cancer cells. MethodDEN combined with CCl4 was used to construct a mouse model of liver cancer via intraperitoneal injection. A normal group， a model group， and a QZAP group were set up， in which QZAP （3.51 g·kg-1） or an equal volume of normal saline was administered daily by gavage， respectively. Serum and liver samples were collected after eight weeks of intervention. Serum alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， γ-glutamyltransferase （γ-GT）， and alpha-fetoprotein （AFP） in mice were detected to evaluate liver function changes of mice in each group. Hematoxylin-eosin （HE） staining and Sirius red staining were used to observe pathological changes in liver tissue. In the cell experiment， Huh7 cells were divided into blank group， QZAP low， medium， and high dose groups and/or PCK1 inhibitor （SKF-34288 hydrochloride） group， and Sorafenib group. The corresponding drug-containing serum and drug treatment were given， respectively. Cell counting kit-8 （CCK-8） method， colony formation experiment， Edu fluorescent labeling detection， intracellular adenosine triphosphate （ATP） content detection， and cell cycle flow cytometry detection were used to evaluate the proliferation ability， energy metabolism changes， and change in the cell cycle of Huh7 cells in each group. Western blot was used to detect the protein expression levels of PCK1， serine/threonine kinase （Akt）， phosphorylated Akt （p-Akt）， and cell cycle-dependent protein kinase inhibitor 1A （p21）. ResultCompared with the model group， the pathological changes such as cell atypia， necrosis， and collagen fiber deposition in liver cancer tissue of mice in the QZAP group were alleviated， and the number of liver tumors was reduced （P<0.01）. The serum ALT， AST， γ-GT， and AFP levels were reduced （P<0.01）. At the cell level， compared with the blank group， low， medium， and high-dose groups of QZAP-containing serum and the Sorafenib group could significantly reduce the survival rate of Huh7 cells （P<0.01） and the number of positive cells with Edu labeling （P<0.01） and inhibit clonal proliferation ability （P<0.01）. The QZAP groups could also reduce the intracellular ATP content （P<0.05） and increase the distribution ratio of the G0/G1 phase of the cell cycle （P<0.05） in a dose-dependent manner. Compared with the model group and blank group， PCK1 and p21 protein levels of mouse liver cancer tissue and Huh7 cells in the QZAP groups were significantly reduced （P<0.05，P<0.01）， and the p-Akt protein level was significantly increased （P<0.01）. Compared with the blank group， the ATP content and cell survival rate of Huh7 cells in the SKF-34288 hydrochloride group were significantly increased （P<0.05）， but there was no statistical difference in the ratio of Edu-positive cells and the proportion of G0/G1 phase distribution. Compared with the SKF-34288 hydrochloride group， the QZAP combined with the SKF-34288 hydrochloride group significantly reduced the ATP content， cell survival rate， and Edu-positive cell ratio of Huh7 cells （P<0.05） and significantly increased the G0/G1 phase distribution proportion （P<0.05）. ConclusionQZAP may induce the metabolic reprogramming of liver cancer cells by activating PCK1 to promote Akt/p21-mediated tumor suppression， thereby exerting an anti-hepatocellular carcinoma proliferation mechanism.
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AIM To study the effects of Biejiajian Pills (Colla Carapacis Trionycis,Asini Corii Colla,Nidus Vespae,etc.) on NF-κB,p65,p50 and IκB in NF-κB signaling pathway and target gene expression in HSC-T6 cells of rats.METHODS HSC-T6 cells were cultured with Biejiajian Pills drug serum for 24 hours,the expressions of p65,p50,VEGF and TIMP-1 mRNA were determined by qPCR;the expression of p65 was measured by immunofluorescence;the expressions of IκBα,IκBβ and α-SMA were determined by Western blot.RESULTS The Biejiajian Pills middle-,high-dose and positive control groups showed significantly lower expressions of p65,VEGF and TIMP-1 mRNA as compared with the blank control group and negative control group,the expressions of p50 mRNA among various groups showed no significant differences.But immunofluorescence showed that the expression of p65 in cytoplasm was decreased.Meanwhile,Biejiajian Pills showed significantly higher IκBα protein expression and obvious down-regulation of α-SMA expression in a dose-dependent manner,but had no significant influence on the expression of IκBβ.CONCLUSION Biejiajian Pills' therapeutic effects on hepatic fibrosis may be related to influencing NF-κB signaling pathway and inhibiting the expression of down-stream target gene.
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Hepatocellular carcinoma(HCC)is one of the most common malignancies worldwide.Due to the difficulty of diagnosis in the early stage of HCC, most HCCs are diagnosed in intermediate-advanced stage.Moreover, the high invasion, metastasis and recurrence rate of HCC result in the high mortality of HCC.MicroRNAs(miRNAs) are a class of highly conserved, endogenous, small, non-coding ,single stranded RNA with the length of 22 nucleotides.There are plentiful of miRNAs in liver.MiRNAs not only can regulate the growth and development of liver, but also are closely related to the formation of HCC.In the process of HCC formation, miRNAs could function as oncogenes or tumor suppressor genes to regulate multiple biological processes related to HCC, including cell differentiation,proliferation,tumorigenesis,angiogenesis,invasion,and metastasis.With the intensive study of molecular mechanisms of miRNAs in the process of HCC formation, increasingly studies have revealed that miRNAs could become sensitive biomarkers and effective therapeutic targets for HCC.
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The present article reports the method of preparation of epidermal growth factor (EGF) from mouse submaxillary glands by using a two-step liquid chromatography and the efficacy of EGF in promoting animal wound healing. It was found that combined purification of crude EGF is better with Bio-gel p-10 and DEAE-DE52 ion-exchange chromatographies and purified EGF has a marked enhancement of wound healing and reduction of cicatricial contracture in animals.
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In this study collagen was applied locally for the treatment of burn wound in the rat. Twenty rats weighing 18-22g were made into 7% [1 degree burn model and divided into two groups: collagen group and control group. After burn, collagen (0.4ml, once daily) was smeared on the rat wound in the treatment group, whereas saline (0.4ml, once daily) in the control group. It was found that collagen can significantly accelerate burn wound healing by comparing the wound healing time and histological changes.
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Microskin autografts were covered with artificial collagen skin, pig skin and cadaver skin in the rats. The results showed that the adhesion to wound and hemostasis of artificial collagen skin was better than pig or cadaver skins. The histological section of healing wound with artificial collagen skin showed that there were revascularization and fibroblast infiltration. The results suggest that artificial collagen skin may be used instead of allograft.