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This study aimed to investigate the relationship between coagulating cold and blood stasis syndrome and glycolysis, and observe the intervention effect of Liangfang Wenjing Decoction(LFWJD) on the expression of key glycolytic enzymes in the uterus and ovaries of rats with coagulating cold and blood stasis. The rat model of coagulating cold and blood stasis syndrome was established by ice-water bath. After modeling, the quantitative scoring of symptoms were performed, and according to the scoring results, the rats were randomly divided into a model group and LFWJD low-, medium-and high-dose groups(4.7, 9.4, 18.8 g·kg~(-1)·d~(-1)), with 10 in each group. Another 10 rats were selected as the blank group. After 4 weeks of continuous administration by gavage, the quantitative scoring of symptoms was repeated. Laser speckle flowgraphy was used to detect the changes of microcirculation in the ears and uterus of rats in each group. Hematoxylin-eosin(HE) staining was used to observe the pathological morphology of uterus and ovaries of rats in each group. The mRNA and protein expressions of pyruvate dehydrogenase kinase 1(PDK1), hexokinase 2(HK2) and lactate dehydrogenase A(LDHA) in the uterus and ovaries of rats were examined by real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot, respectively. The rats in the model group showed signs of coagulating cold and blood stasis syndrome, such as curl-up, less movement, thickened veins under the tongue, and reduced blood perfusion in the microcirculation of the ears and uterus, and HE staining revealed a thinning of the endometrium with disorganized arrangement of epithelial cells and a decrease in the number of ovarian follicles. Compared with the model group, the treatment groups had alleviated coagulating cold and blood stasis, which was manifested as red tongue, reduced nail swelling, no blood stasis at the tail end as well as increased blood perfusion of the microcirculation in the ears and uterus(P<0.05 or P<0.01). Among the groups, the LFWJD medium-and high-dose groups had the most significant improvement in coagulating cold and blood stasis, with neatly arranged columnar epithelial cells in uterus, and the number of ovarian follicles was higher than that in the model group, especially mature follicles. The mRNA and protein expressions of PDK1, HK2, LDHA in uterus and ovaries were up-regulated in the model group(P<0.05 or P<0.01), while down-regulated in LFWJD medium-and high-dose groups(P<0.05 or P<0.01). The LFWJD low-dose group presented a decrease in the mRNA expressions of PDK1, HK2 and LDHA in uterus and ovaries as well as in the protein expressions of HK2 and LDHA in uterus and HK2 and PDK1 in ovaries(P<0.05 or P<0.01). The therapeutic mechanism of LFWJD against coagulating cold and blood stasis syndrome is related to the down-regulation of key glycolytic enzymes PDK1, HK2 and LDHA, and the inhibition of glycolytic activities in uterus and ovaries.
Subject(s)
Female , Animals , Rats , Ovary , Uterus , Ovarian Follicle , Lactate Dehydrogenase 5 , GlycolysisABSTRACT
Objective::To study the effect of Qingzao Jiufei Tang on the expression of key limiting enzymes hexokinase 2(HK2), phosphofructokinase 2(PFK2) and pyruvate kinase M2 (PKM2), and the glucose content in Lewis mice colon cancer cells. Method::A total of 50 male C57BL/6J mice were randomly divided into model group, chemotherapy group, and high, middle and low-dose Qingzao Jiufei Tang groups, with 10 mice in each group. The lung cancer cell model was established by injecting Lewis lung cancer cells into the right axilla. The high, middle and low dose groups were administered at the doses of 11, 5.5, 2.75 g·kg-1·d-1 for 2 weeks before modeling. The drug was administered through intraperitoneal injection at a dose of 50 mg·kg-1·(2 d)-1 in the chemotherapy group. The model group was intragastrically administered with an equal volume of normal saline. After the inoculation, the drug was administered for two weeks. Two weeks later, all of the mice were put to death, and tumor tissues were collected. The mRNA expression of HK2 was detected by Real-time PCR. the protein expression of PFK2 was detected by Western blot, the PKM2 activity was detected by enzyme-linked immunosorbent assay (ELISA). Result::Compared with the model group, mRNA expressions and activity of PKM2 in lung cancer cells of treatment groups were significantly declined, and glucose content increased significantly, with significant differences from those of model group (P<0.01). The PFK2 protein expressions in lung cancer cells of treatment groups (high, medium and low-dose groups) were significantly decreased (P<0.05, P<0.01). Conclusion::Qingzao Jiufei Tang could inhibit Lewis proliferation, and decrease the glucose intake in lung cancer cells. The effect targets may be the key rate-limiting enzymes HK2, PFK2, PKM2.
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Objective To analyze the association between hexokinase 2 (HK2) gene expression and clinical pathological characteristics in renal clear cell carcinoma using database and bioinformatics methods. Methods Oncomine database was used to analyze the most significant differentially expressed genes between renal clear cell carcinoma and non-kidney tissue. The expression levels of mRNA and protein were detected by GPEIA and The Human Protein Atlas database. Correlation of HK2 expression level between clinicopathological features and prognosis of renal clear cell carcinoma was analyzed using LinkedOmics and KM-plotter databases, respectively. The STRING database was used to predict the potential protein interaction mechanism. Results The most significant difference protein in renal clear cell carcinoma, i.e. HK2, was found, which was highly expressed in renal clear cell carcinoma tissues, and positively correlated with pathological stage, T stage and N stage of renal cell carcinoma (all P<0.05). The overall survival rate of the renal clear cell carcinoma patients with high expression of HK2 was significantly lower than that of the patients with low expression (P<0.05). Conclusions High expression of HK2 gene may be associated with pathological staging, high T stage, high N stage, and poor prognosis of renal clear cell carcinoma.
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Objective: To explore the expression of hexokinase 2 (HK2) in ovarian cancer tissues and its effect on the proliferation of ovarian cancer cells. Methods: The levels of HK2 in cancer tissues of 27 patients and normal tissues of 14 patients were detected with qRT-PCR and immunohistochemical analysis. Overexpression of HK2 by transfected PLHKII-pGFPN3 plasmid and knockdown of HK2 by transfected HK2 siRNA were used in our study. Proliferation of SKOV3 cells was detected by CCK8 method. The expression of HK2 was measured by Western blot. Results: The expression of HK2 in 27 patients with ovarian cancer (92.6%, 25/27) was up-regulated compared with normal tissues (42.9%, 6/14). The expression of HK2 in the cancer tissues of patients with clinical stage III-, pathological grade G3, and distant metastasis was up-regulated significantly (P<0.05). The growth of SKOV3 cells was significantly promoted by overexpression of HK2, and the growth of SKOV3 cells was inhibited by downexpression of HK2. Conclusion: The expression of HK2 is significantly higher in ovarian cancer tissues than in normal ovarian tissues. HK2 can promote the proliferation of ovarian cancer cells.
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Objective@#To analyze the association between hexokinase 2 (HK2) gene expression and clinical pathological characteristics in renal clear cell carcinoma using database and bioinformatics methods.@*Methods@#Oncomine database was used to analyze the most significant differentially expressed genes between renal clear cell carcinoma and non-kidney tissue. The expression levels of mRNA and protein were detected by GPEIA and The Human Protein Atlas database. Correlation of HK2 expression level between clinicopathological features and prognosis of renal clear cell carcinoma was analyzed using LinkedOmics and KM-plotter databases, respectively. The STRING database was used to predict the potential protein interaction mechanism.@*Results@#The most significant difference protein in renal clear cell carcinoma, i.e. HK2, was found, which was highly expressed in renal clear cell carcinoma tissues, and positively correlated with pathological stage, T stage and N stage of renal cell carcinoma (all P<0.05). The overall survival rate of the renal clear cell carcinoma patients with high expression of HK2 was significantly lower than that of the patients with low expression (P<0.05).@*Conclusions@#High expression of HK2 gene may be associated with pathological staging, high T stage, high N stage, and poor prognosis of renal clear cell carcinoma.