Effect of DNA methylation of miR-30a-5p promoter region on hepatic injury induced by homocysteine

AIM: To explore the role of DNA methylation of microRNA-30a-5p(miR-30a-5p) promoter region in hepatic injury. METHODS: Four-week-old normal mice and cystathionine β-synthase (CBS) single gene knockout mice were used and divided into normal (CBS^+/+, n=12) group and single gene knockout (CBS^+/-, n=12) group, and the mice were fed with high methionine diet for 8 weeks. HL-7702 hepatic cells were routinely cultured in vitro and divided into control group, homocysteine (Hcy) group and Hcy+5-azacytidne (AZC) group. Serum Hcy, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured by automatic biochemical analyzer. The levels of ALT and AST in the cells culture medium were determined by the microplate method. Hepatic injury in the mice were observed with HE staining. Cell viability staining was used to measure the viability of hepatocytes. RT-qPCR was used to detect the expression of miR-30a-5p in the liver tissues and hepatocytes. The correlation between the expression of miR-30a-5p and serum ALT and AST levels was analyzed by Pearson correlation analysis. DNA methylation level of miR-30a-5p promoter region in the liver tissues and hepatocytes was detected by nested landing methylation-specific PCR (nMS-PCR). RESULTS: Compared with the CBS^+/+ mice, the serum levels of Hcy, ALT and AST in the CBS^+/- mice were significantly increased (P < 0.05). HE staining showed the hepatocyte swelling and nuclear fragmentation and dissolution. The expression level of miR-30a-5p in the liver tissues was decreased (P < 0.01). Besides, the expression level of miR-30a-5p in the mice was negatively correlated with serum ALT and AST levels (r²=0.4557, P=0.0003, r²=0.4626, P=0.0003), and the DNA methylation of miR-30a-5p promoter region was increased (P < 0.01). In the HL-7702 cells, compared with control group,the ALT and AST levels were increased in Hcy group (P < 0.05, P < 0.01), and the cell viability was remarkablely decreased. DNA methylation of miR-30a-5p promoter region was increased (P < 0.01), which decreased after treated the cells with AZC (P < 0.05), while the expression level of miR-30a-5p in the cells was increased (P < 0.05). CONCLUSION: Hypermethylation of miR-30a-5p promoter region may play an important role in hepatic injury.     

Effect of miR-29b-mediated TGF-β/Smad signaling pathway on progression of hepatic fibrosis in rats

AIM: To investigate the role of microRNA-29b (miR-29b)-mediated TGF-β/Smad signaling pathway in the activation of hepatic stellate cells (HSC) and its effect on the progression of hepatic fibrosis in rats.METHODS: Hepatic liver fibrosis rat model was established, and its HSC were isolated. Normal rat HSC were also obtained and identified in vitro. RT-qPCR and Western blot were used to detect the alterations of miR-29b, TGF-β/Smad signaling pathway-related proteins and liver fibrosis marker proteins in the acquired cells. Finally, the direct targeting binding of miR-29b to TGF-β1 was identified by dual-luciferase reporter assay system.RESULTS: With the activation of HSC, the expression of miR-29b gradually decreased (P<0.01), while the expression of collagen type I and α-smooth muscle actin gradually increased (P<0.01). At the same time, the expression of Smad2/3/4 was significantly increased, and the expression of Smad7 was significantly decreased (P<0.01). Dual-luciferase reporter assay showed that miR-29b bound directly to "UCUCUCCGU" in the 3'UTR of TGF-β1, indicating that TGF-β1 was a downstream target gene of miR-29b.CONCLUSION: miR-29b may be involved in the inhibition of HSC activation and migration, thereby inhibiting the process of liver fibrosis. The biological function of miR-29b may be through the direct targeting of TGF-β1, thus regulating and inhibiting the TGF-β/Smad signaling pathway.     

Altered pharmacokinetics of enrofloxacin in experimental models of hepatic and renal impairment

We investigated the effects of hepatic and renal impairment on the pharmacokinetics of enrofloxacin in Sprague-Dawley rats. Experimental hepatic and renal failure were induced by carbon tetrachloride (CCL₄) and 5/6 nephrectomy, respectively. After intravenous dosing of enrofloxacin (10 mg/kg), plasma concentrations of enrofloxacin were measured using liquid chromatograph/mass spectrometry. There was no significant effect of hepatic impairment on enrofloxacin pharmacokinetics. However, renal impairment markedly prolonged elimination half life (t_1/2λz) of enrofloxacin (P < 0.05), comparing with respective control. Total body clearance (Cl_b) and volume of distribution at steady state (V_ss) were significantly decreased (P < 0.05) by renal impairment. In conclusion, these results suggested that renal impairment could affect the pharmacokinetics of enrofloxacin.     

Effect of siRNA-mediated Smad3 silence on proliferation and apoptosis in activated hepatic stellate cells

AIM: To investigate the effects of siRNA-mediated Smad3 silence on proliferation and apoptosis in activated hepatic stellate cells (HSCs).METHODS: HSCs-T6 cells were divided into 3 groups: blank group, negative control group and siRNA-Smad3 transfection group. The siRNA-Smad3 was transfected into HSCs-T6 cells. At different time points after transfection, cell proliferation was measured by CCK-8, cell apoptosis was detected by flow cytometry, and protein levels of P53 and Bcl-2 were determined by immunocytochemistry.RESULTS: HSCs proliferation was significantly inhibited at the time points of 24 h, 48 h and 72 h after transfection. Meanwhile, the apoptosis of HSCs was significantly increased in siRNA-Smad3 transfection group (P<0.01). Compared to the control cells, the protein expression of P53 was significantly increased while Bcl-2 protein was significantly decreased 48 h after transfection in siRNA-Smad3 transfection group (P<0.01).CONCLUSION: The siRNA-mediated Smad3 silence significantly inhibits HSCs proliferation and induces apoptosis by up-regulating the P53 expression and down-regulating the Bcl-2 expression in HSCs.     

鸡马杜霉素急性中毒及其脂质过氧化关系的研究

本文在实验条件下人工诱发鸡马杜霉素急性中毒，观察中毒症状及病理变化，测定受损靶器官的脂质过氧化物的含量及相关酶的活性，以及肝细胞色素Ｐ－４５０的含量。实验结果表明：鸡马杜霉素急性中毒的主要临床症状是腹泻、腿无力或麻痹；肉眼病变是全身性充血、淤血及少数组织出血；显微镜检病变是肝脏、心肌及腿肌出血与变性；肝脏的脂质过氧化物的含量及过氧化氢酶的活性显著增加；肝Ｐ－４５０的含量显著增加；但是心肌与腿肌的脂     

高产乳牛脂肪肝性肝障碍的某些生化成分的分析

选用黑白花牛31头(健康初产和经产牛各5头,病牛21头),监测其产犊前后血清脂质、蛋白质、酶、血糖等值的动态变化。阐明初产牛患脂肪肝病的少于经产牛,因其产犊前脂肪动员量低于经产牛,同时被动员的甘油三脂(TG)与游离脂肪酸(FFA)的血清浓度呈负相关。 预产前7～10天,健康经产牛的白蛋白(Al)和白蛋白与球蛋白之比值(A/G)为50％和1左右。谷草转氨酶(GOT)和乳酸脱氢酶总酶(LDH),分别为38±6.Ou和870±67.8u。其LDH同工酶值呈LDHI>LDH_2>LDH_3>LDH_4>LDH_5,均稳定在正常范围。而疑有肝障碍病牛经治愈的17头,初诊时Al和A/G值已分别下降至29.8±7.6％和0.42±0.22,GOT和LDH值均上升,血搪(Glu)值下降,LDH_5倒置(LDH_1>LDH_2>LDH_3>LDH_4LDH_2>LDH_5>LDH_3>LDH_4％)。     

Assessment of the Efficacy and Potential Complications of Transjugular Liver Biopsy in Canine Cadavers

Background

Transjugular liver biopsy (TJLB) is used in humans at risk of bleeding. There are no reports of its use in veterinary medicine.

Objective

To assess the efficacy and potential complications of TJLB in canine cadavers, and compare with samples obtained via needle liver biopsy (NLB) and surgical liver biopsy (SLB).

Animals

Twenty‐five medium and large breed canine cadavers.

Methods

Prospective study. TJLBs were procured through the right jugular vein. After biopsy, intravenous contrast and gross inspection were used to assess the biopsy site. Minor and major complications were recorded. NLBs and SLBs were then obtained. Histopathology was performed, and TJLB and NLB were compared for number of complete portal tracts (CPTs), length, and fragmentation. Pathologic process and autolysis were assessed in all samples.

Results

All TJLBs yielded liver tissue. The proportion of minor complications was 12/25 (48%), and major complications 16/25 (64%); 13/16 (81%) of the major complications were liver capsule perforation. In 21/25 (84%), the histopathology in the SLB was reflected in the TJLBs. For cases with minimal autolysis, median number of CPTs in TJLBs was 7.5, compared with 4 in NLBs (P = .018). Median length of TJLB specimen was 28 mm compared to 22 mm in NLBs (P = .007). Fragmentation rate was median of 1.25 for TJLB compared to 1.50 in NLBs (P = .11).

Conclusions and Clinical Importance

TJLB is technically feasible and achieves comparable results to NLB and SLB. The number of complications, in particular liver capsule perforation, was greater than expected. Further studies are indicated before clinical use is recommended.     

五杞胶囊对大鼠实验性肝纤维化的保护作用

目的观察五杞胶囊对大鼠实验性肝纤维化的保护作用。方法 Wistar大鼠随机分为对照组、模型组、阳性药物组及五杞胶囊2、4、8g生药/kg组。皮下注射CCL4花生油溶液10周建立大鼠实验性肝纤维化模型,五杞胶囊给药4周后观察大鼠肝脏系数,测定血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、胆碱酯酶(CHE)活力及白蛋白(ALB)、球蛋白(GLO)、总蛋白(TP)含量,肝组织超氧化物歧化酶(SOD)活性及丙二醛(MDA)、羟脯氨酸(Hyp)含量。结果五杞胶囊能明显降低大鼠肝脏系数,升高大鼠血清ALB含量及A/G比值,降低血清AST、ALT、CHE活力及GLO含量,减少肝组织Hyp、MDA含量,升高SOD活性。结论五杞胶囊通过减轻肝细胞脂质过氧化损伤改善肝功能,纠正肝纤维化引起的白蛋白降低。