The cerebral GABA-T activity of mice was determined by the modified coupled-enzyme spectroflourimctry, and the method was proved stable and feasible. The cerebral GABA degradation of mice in vitro was inhibited by the sera from the mice with hepatic coma induced by galactosamine. The supernatant of liver homogenate of both control and hepatic coma groups can inhibit the cerebral GABA degradation too. The inhibitory capacity is proportional to the volume of LHS added in certain conditions, and reaches a maximum inhibitory rate of 80%. The inhibition can not be relieved by pyridoxal phosphate.The inhibitory effector of cerebral GABA degradation has been regarded as liver specific or mainly presented in the liver since no appreciable inhibition was found after the addition of the supernaiants of kidney, heart, lung and spleen homogenates. The possible contribution of GABA degradation inhibition by LHS to the pathogenesis of hepatic encephalopathy has also been discussed

Objective:To investigate the effects of microRNA (miR)-513a-3p regulating hexokinase 2 (HK2) on proliferation and glycometabolism in colorectal cancer cell.Methods:From May 2019 to February 2020, the miR-513a-3p simulant, miR-513a-3p inhibitor and miR control were transfected into colorectal cancer SW480 cell respectively. Real-time quantitative polymerase chain reaction was used to detect the expression levels of miR-513a-3p in colorectal cancer SW480 cell, normal colorectal cell and all transfected colorectal cancer SW480 cell. The effect of miR-513a-3p on cell proliferation was detected by CCK-8 assay. Brd/PI incorporation assay was used to detect the effect of miR-513a-3p on glycometabolism. Western blot was used to detect the expression of HK2.Results:The expression level of miR-513a-3p in colorectal cancer SW480 cell was significantly lower than that in normal colorectal cell (0.43 ± 0.06 vs. 1.00 ± 0.02), and there was statistical difference ( t = 7.024, P = 0.003). The expression level of miR-513a-3p in colorectal cancer SW480 cell transfected with miR-513a-3p simulant was significantly higher than that in colorectal cancer SW480 cell transfected with miR control and transfected with miR-513a-3p inhibitor (1.18 ± 0.24 vs. 0.45 ± 0.04 and 0.22 ± 0.03), the expression level of miR-513a-3p in colorectal cancer SW480 cell transfected with miR control was significantly higher than that in colorectal cancer SW480 cell transfected with miR-513a-3p inhibitor, and there were statistical differences ( P<0.05). The proliferation ability in colorectal cancer SW480 cell transfected with miR-513a-3p simulant at 24, 48, 72 and 96 h was significantly lower than that in colorectal cancer SW480 cell transfected with miR-513a-3p control group, the proliferation ability in colorectal cancer SW480 cell transfected with miR-513a-3p inhibitor at 24, 48, 72 and 96 h was significantly higher than that in colorectal cancer SW480 cell transfected with miR-513a-3p control, and there were statistical differences ( P<0.05). The glucose intake, lactate and thioredoxin-interacting protein (TXNIP) expression levels in colorectal cancer SW480 cell transfected with stimulant were significantly lower than those in colorectal cancer SW480 cell transfected with miR control (1.02 ± 0.04 vs. 1.90 ± 0.06, 0.88 ± 0.03 vs. 1.45 ± 0.04 and 0.16 ± 0.02 vs. 0.86 ± 0.06), the indexes in colorectal cancer SW480 cell transfected with miR-513a-3p inhibitor were significantly higher than those in colorectal cancer SW480 cell transfected with miR control (2.35 ± 0.09 vs. 1.90 ± 0.06, 1.67 ± 0.08 vs. 1.45 ± 0.04 and 2.01 ± 0.15 vs. 0.86 ± 0.06), and there were statistical differences ( P<0.05). The expression level of HK2 in colorectal cancer SW480 cell transfected with miR-513a-3p stimulant was significantly lower than that in colorectal cancer SW480 cell transfected with miR control (0.20 ± 0.01 vs. 1.02 ± 0.04), and there was statistical difference ( t = 8.367, P<0.05); the expression level of HK2 in colorectal cancer SW480 cell transfected with miR-513a-3p inhibitor was significantly higher than that in colorectal cancer SW480 cell transfected with miR control (1.91 ± 0.07 vs. 1.02 ± 0.04), and there was statistical difference ( t = 4.279, P<0.05). Conclusions:MiR-513a-3p can significantly inhibit the proliferation and glycometabolism of colorectal cancer cell, and its regulatory mechanism is related to the inhibition of the HK2 protein expression in the cell by miR-513a-3p.

Objective:To investigate the relationship between the expression of inhibin subunit Beta A (INHBA) and the clinicopathological data and its effect on proliferation, invasion and metastasis of gastric cancer cells and its possible mechanisms.Methods:Using Gene Expression Profiling Interactive Analysis (GEPIA) public database to verify the expression of INHBA mRNA in gastric cancer and adjacent tissues and its relationship with pathological stage and prognosis; the relationship between INHBA and clinical prognosis was analyzed using the Kaplan-Meier Plotter online database. Immunohistochemistry was used to confirm the correlation between protein expression level of INHBA in gastric cancer and adjacent tissues and clinical pathological staging. In vitro, the cell proliferation was detected by tetrazolium salt (MTT) method; the cell migration ability was detected by scratch test, and cell invasion and metastasis ability was detected by transwell chamber assay. The expression of INHBA and epithelial-mesenchymal transition (EMT) related proteins was detected by Western blot. Results:The GEPIA database and Kaplan-Meier Plotter online database analysis showed that INHBA mRNA was highly expressed in various cancer tissues and significantly higher in gastric cancer tissues than normal tissues ( P<0.05). The high expression of INHBA mRNA was associated with clinical stage and poor prognosis of gastric cancer ( P<0.05). The results of immunohistochemistry showed that the staining score of INHBA in gastric cancer tissues was significantly higher than that in adjacent tissues ( P<0.05), and its expression level was correlated with clinical tumor node metastasis (TNM) stage ( P<0.05). The results of MTT, scratch test and transwell chamber showed that INHBA overexpression could promote the proliferation, migration, invasion and metastasis of gastric cancer cells, while interference with INHBA expression could inhibit the proliferation, migration, invasion and metastasis of gastric cancer cells; Western blot results showed that the expression of CDH1 was down regulated and the expression of CDH2 was up-regulated after INHBA overexpression. The expression of CDH1 was up-regulated and the expression of CDH2 was down-regulated after INHBA overexpression was inhibited. Conclusions:INHBA is highly expressed in gastric cancer tissues compared with adjacent tissues. The expression level of INHBA is related to tumor progression and poor prognosis. INHBA can promote the proliferation, migration and invasion of gastric cancer MGC-803 cell line and its mechanism may be related to INHBA promoting cell EMT.

Objective:To investigate the expression and clinical significance of growth differentiation factor 15 (GDF-15) in papillary thyroid carcinoma (PTC).Methods:The tumor tissues and metastatic lymph node tissues of 3 PTC patients who underwent radical surgery of thyroid cancer in the First Hospital of Hunan University of Chinese Medicine from January to February 2019 were collected, and the differential expressed genes were screened by high-throughput sequencing; 20 cases of primary tumor tissues and metastatic lymph node tissues were collected to verify the sequencing results. Another 20 cases of primary PTC tissues and adjacent tissues (>2 cm away from the tumor edge) were collected to verify the expression of target genes in tumor tissues and adjacent tissues. Sixty-four pathological specimens of PTC patients who underwent radical surgery of thyroid cancer in the First Hospital of Hunan University of Chinese Medicine from January to December 2014 were collected, of which 31 patients had lymph node metastasis. The real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of GDF-15 and verify the sequencing results; immunohistochemistry was used to detect the expression of GDF-15 protein in the primary PTC tissues, adjacent tissues and metastatic lymph node tissues. According to the expression of GDF-15 protein in the primary tumor tissues of PTC patients, the patients were divided into high-expression group (35 cases) and low-expression group (29 cases), and the relationship between GDF-15 expression level and clinicopathological characteristics of patients was analyzed; Kaplan-Meier method was used to analyze the 5-year tumor-free survival rate of the two groups.Results:The mRNA high-throughput sequencing results of 3 cases of PTC primary and metastatic tissues showed that the top 10 differential expressed genes were CDH2, CDF15, DKK1, GLIPR1, PCDH7, ID3, FBN1, MYPN, UBASH3B and CCDC80. The expression of GDF-15 mRNA in 20 cases of PTC primary tumor tissues and adjacent tissues were 4.1±0.5 and 2.8±0.3, and the difference was statistically significant ( t = 2.220, P = 0.032). The expression of GDF-15 mRNA in another 20 cases of PTC primary tumor tissues and metastatic lymph node tissues were 3.1±0.4 and 5.8±0.7, and the difference was statistically significant ( t = 3.556, P = 0.001). The results of immunohistochemistry showed that GDF-15 had the immunohistochemical scores of (4.0±0.3) points, (6.1±0.3) points and (9.0±0.4) points in PTC adjacent tissues, primary tumor tissues and metastasis tissues. The expressions of GDF-15 protein between PTC primary tumor tissues and adjacent tissues, metastatic tissues and adjacent tissues, and metastatic tissues and primary tumor tissues were significantly different (all P < 0.01). The differences in composition ratios of tumor long-axis diameter, tumor T stage and N stage between GDF-15 high-expression group and low-expression group were statistically significant (all P<0.05). The 5-year tumor-free survival rates in GDF-15 high-expression group and low-expression group were 60% and 83%, and the difference was statistically significant ( P = 0.033). Conclusions:The expressions of GDF-15 in PTC adjacent tissues, tumor tissues and metastatic lymph node tissues gradually increase, and its expression level is related to tumor progression, recurrence and metastasis. It can be used as a potential clinical prognostic warning molecule and therapeutic target.

Background Dry eye is increasing gradually recently,but its etiology and manifestation are very diverse.Studies showed that menopause of adult females was one of the risk factors of dry eye.In addition,some inflammatory factors also participate in the pathogenesis and development.But the study on the relationship of sex hormone with inflammation and ocular surface damage is still below understanding.Objective This study was to investigate the expressing changes of interleukin (IL) and tumor necrosis factor-α (TNF-α) in conjunctiva and the manifestation of ocular surface in ovariectomized rat model.Methods Twenty clear female SD rats were randomized into the ovariectomized group and the sham operative group according to randomized number table.Ovariectomy was performed in the ovariectomized group,and abdominal myotomy without ovariectomy was performed in the sham operative group.Serum estrogen and androgen levels were detected by radiation immunoassay 3 months after operation.Schirmer Ⅰ test (S Ⅰ t) and corneal fluorescence staining were carried out in the rats before operation and 1 month,2 and 3 months after operation.The morphology of conjunctival epithelial cells was examined by hematoxylin & eosin staining at the 3rd month after operation.The expressions of IL-17A,IL-1 β,IL-6 and TNF-α in conjunctiva were semi-quantitative analyzed by immunohistochemistry and Western blot.The use and care of the animals complied with State Science and Technology Commission Regulations for the Administration of Affair Concerning Experimental Animals.Results Serum estrogen levels were (23.53 ± 1.65) pg/L and (47.89 ± 1.05) pg/L 3 months after surgery in the ovariectomized group and the sham operative group,respectively; the serum androgen levels were (1.84±0.09) ng/L and (2.47±0.12)ng/L in the ovariectomized group and the sham operative group,respectively,showing a significant decline of serum estrogen and androgen levels in the ovariectomized group compared with the sham operative group (t=-35.37,-12.13,both at P＜0.01).No significant differences were seen in S Ⅰ t between the two groups among various time points (Fgroup =0.38,P =0.55 ; Ftime =0.13,P =0.72 ; Finteraction =0.39,P =0.76).No obvious fluorescence staining was seen in the cornea of both the ovariectomized group and the sham operative group.The histopathological examination showed that the layers of rat conjunctival epithelial cells increased with the disordered arrangement in the ovariectomized group.Immunochemistry showed that the expressions of IL-17A,IL-1β,IL-6 and TNF-α (A values) were significantly higher in the ovariectomized group than those in the sham operative group (IL-17A:t=8.22,P＜0.01 ;IL-1β:t=16.43,P＜0.01 ;IL-6:t=13.44,P＜0.01 ;TNF-α:t=16.26,P＜0.01).Western blot assay showed the similar results (IL-17A:t=19.41,P＜0.01 ;IL-1β:t=12.63,P＜0.01 ;IL-6:t=17.17,P＜0.01 ;TNF-α:t=15.19,P＜0.01).Conclusions Serum estrogen and androgen levels drop obviously,and there is an up-regulation of IL and TNF-α expression in conjunctiva tissue in the ovariectromized SD rats.However,no obvious dry eye-related sign occurs.

Background Retinal Muller cells participate in the pathological process of diabetic retinopathy (DR) through expressing vascular endothelial growth factor (VEGF).It is reported that FK506 inhibits the expression of VEGF in solid tumors and experimental corneal neovascularization,but whether FK506 exerts its role on retinal Müller cells or not is still unclear.Objective This study aimed to investigate how FK506 affects the expression vascular endothelial growth factor (VEGF) in rat retinal Müller cells under the condition of high glucose.Methods Immortalized rat retinal Müller cell line was regularly cultivated and logarithmic phase of cells were incubated in 96-well plate with the cell density of 1 × 104/ml.Different concentrations of FK506 (800.00,400.00,200.00,100.00,75.00,50.00,25.00,12.50 and 6.25 pg/ml) (100 μl/well) were added into the culture medium to determine the half maximal inhibitory concentration (IC50) of FK506 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).The cell lines were cultured with DMEM medium (containing D-glucose of 5.5 mmol/L) or high glucose DMEM (containing D-glucose of 50 mmol/L),and 75 pg/ml FK506 were added into DMEM,respectively,and the cells were divided into the normal control group,FK506 group,high glucose culture group and high glucose + FK506 group.ELISA was employed to assay the content of VEGF protein in the cell supernatant.The expressions of VEGF mRNA and protein in the cells were detected by reverse transcription PCR (RT-PCR) and Western blot,respectively.Results The cells grew well in the normal control group,FK506 group,high glucose culture group and high glucose+FK506 group in 12,24 and 48 hours after culture with the polygon-like shape.The IC50 of FK506 was 75 pg/ml.The contents of FK506 in the cell supernatant were (966.46± 13.59) pg/ml,(1 059.42±67.43) pg/ml,(16 243.11 ±3 926.38) pg/ml and (9 467.25± 1 525.56) pg/ml in the normal control group,FK506 group,high glucose culture group and high glucose+FK506 group,respectively,showing a significant difference among the four groups (F =20.51,P =0.00).The VEGF levels in cell supernatant were significantly higher in the high glucose group than those of the normal group and the high FK506 group (P =0.00,P =0.02),but no significant difference was found in the VEGF level in cell supernatant between the control group and FK506 group (P =0.08).The expressions of VEGF mRNA and protein in the cells were significantly different among the four groups (F=126.06,P=0.00;F=5.44,P=0.01),and the relative expressing values of VEGF mRNA and protein in the cells of the high glucose group were significantly higher than those of the control group and the high+ FK506 group (all at P＜0.01).The relative expressing values of VEGF mRNA and protein were 0.64±0.09 and 0.68±0.18 in the FK506 group,which were lower than those of the normal control group (0.84±0.07 and 0.75± 0.03).However,no significant differences were seen between the two groups (P =0.05,0.07).Conclusions The expression of VEGF in rat retinal Müller cells up-regulates under the high glucose condition.FK506 plays inhibitory effects on VEGF expression to certain extent in vitro.

Objective:To investigate the expression and clinical significance of oxidized low density lipoprotein receptor 1 (OLR1) in gastric cancer.Methods:In gastric cancer tissues and adjacent tissues, the relationship between the expression of OLR1 and the stage and clinical prognosis of gastric cancer was analyzed from the Gene Expression Profiling Interactive Analysis (GEPIA) database and Kaplan-Meier Plotter online database. We collected 6 case of fresh gastric cancer tissues and adjacent tissues of patients undergoing radical gastrectomy in our hospital from October 2018 to December 2018, The extracted RNA were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) to verify the expression in gastric cancer and adjacent tissues. From January 2012 to January 2014, 36 patients with pathological biopsy specimens of radical gastrectomy in our hospital were examined. Immunohistochemistry was used to confirm the correlation between protein expression level of OLR1 in gastric cancer and paracancerous tissues and clinical pathological stage and prognosis.Results:GEPIA database and Kaplan-Meier Plotter online database analysis showed that OLR1 mRNA was highly expressed in various cancer tissues and significantly higher in gastric cancer tissues than normal tissues ( P<0.05). High expression of OLR1 mRNA is associated with clinical stage and poor prognosis of gastric cancer; qRT-PCR results showed that OLR1 mRNA was highly expressed in gastric cancer tissues; the results of immunohistochemistry showed that the staining score of OLR1 in gastric cancer tissues was significantly higher than that in adjacent tissues ( P<0.001), and its expression level was correlated with depth of tumor invasion and clinical tumor node metastasis (TNM) stage (both P<0.05). Kaplan-Meier survival analysis showed that the expression level of OLR1 was associated with poor prognosis in patients. The median survival time was 18 months in OLR1 high expression group and 30 months in OLR1 low expression group, with statistically significant difference ( P=0.016). Conclusions:OLR1 is highly expressed in gastric cancer tissues, and its expression level is associated with tumor progression and poor prognosis.