The purpose of this study is to evaluate the protective effect of 4-hydroxy-2(3H)-benzoxazolone from Acanthus ilicifolius (HBAI) on acute liver injury induced by acetaminophen in mice and its mechanism. Mice were continuously treated with HBAI (200, 100, 50 mg/kg) once a day for 10 days. After that, the mice were fasted for 8 hours, followed by intraperitoneal injection of acetaminophen (300 mg/kg). The results showed that HBAI pretreatment significantly reduced acetaminophen-induced liver tissue congestion, hepatocyte apoptosis and necrosis, and inflammatory cell infiltration. HBAI could effectively reduce the levels of serum alanine aminotransferase, aspartate aminotransferase, total bilirubin, reactive oxygen species and malondialdehyde. Interestingly, the activities of liver catalase, superoxide dismutase, glutathione and glutathione reductase were enhanced by HBAI pretreatment. Moreover, HBAI pretreatment alleviated acetaminophen-induced hepatocyte apoptosis by regulating the expression of Bcl-2 family proteins and the mitochondrial function. Further study showed that HBAI pretreatment effectively promoted the expression of Nrf2 and its signal downstream HO-1, NQO1, GCLC, GCLM, and MGST-1, suggesting the activation of the Nrf2/HO-1 signaling pathway. Meanwhile, HBAI attenuated the phosphorylation of NF-κBp65, IKKα/β, and IκBα, as well as the expression of NF-κBp50, which indicated that HBAI blocked the signal transduction of NF-κB pathway. In conclusion, HBAI protects against acetaminophen-induced acute liver injury by inhibiting the NF-κB and activating Nrf2/HO-1 signaling pathways.

An ingredient was isolated from Acanthus ilicifolius and identified as 4-hydroxy-2(3H)-benzoxazolone (HBOA). Its protective effects and underlying mechanism on liver fibrosis were investigated. Briefly, rats were intragastrically administrated with 50% CCl4 twice a week for 12 weeks to induce liver fibrosis. Meanwhile, the animals were treated with various medicines from weeks 8 to 12. Then the histological change, serum biochemical index, inflammatory factors and hepatocyte apoptosis were detected. Moreover, the TGF-β1/Smads, NF-κB and ERK signaling pathways were also detected to illustrate the underlying mechanism. The results showed that HBOA significantly ameliorated CCl4-induced liver injury and collagen accumulation in rats, as evidenced by the histopathologic improvement. Moreover, HBOA markedly decreased hepatocyte apoptosis by regulating the expression levels of caspase-3, -9 and -12, as well as the Bcl-2 family. The mechanism study showed that HBOA significantly decreased the expressions of α-smooth muscle actin (α-SMA) and collagen and inhibited the generation of excessive extracellular matrix (ECM) components by restoring the balance between matrix metalloproteinases (MMPs) and its inhibitor (TIMPs). HBOA markedly alleviated oxidative stress and inflammatory cytokines through inhibiting the NF-κB pathway. In addition, HBOA significantly down-regulated the levels of TGF-β1, Smad2/3, Smad4 and up-regulated the level of Smad7, inhibiting the TGF-β1/Smads signaling pathway. Moreover, HBOA significantly blocked the ERK signaling pathway, leading to the inactivation of hepatic stellate cells. This study suggests that HBOA exerts a protective effect against liver fibrosis via modulating the TGF-β1/Smads, NF-κB and ERK signaling pathways, which will be developed as a potential agent for the treatment of liver fibrosis.

Oxidative damage to mitochondrial DNA (mtDNA) and cell apoptosis are heavily implicated in aging. Our previous study established a mimetic rat model of aging in the cochleae using D-galactose (D-gal), and revealed that chronic injection of D-gal can increase oxidative stress and mtDNA common deletions (CD). The aim of the present study was to investigate the sources of reactive oxygen species and the occurrence of apoptosis in the cochleae of rats following 8 weeks of D-gal exposure. The results of the present study indicated that an elevated accumulation of the mtDNA CD and mitochondrial ultrastructural damage occurred in the cochleae of rats injected with D-gal for 8 weeks. In addition, the levels of 8-hydroxy-2-deoxyguanosine, NADPH oxidase (NOX) 3, P22(phox) and cleaved caspase 3, and the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end-labelling-positive cells were increased in the cochleae of D-gal-treated rats, compared with the controls. These findings suggested that nitric oxide synthase NOX3-associated oxidative stress may contribute to the accumulation of mtDNA mutations and activate a caspase 3-dependent apoptotic signalling pathway in the cochleae during aging. The present study also provided novel insights into the development of age-associated hearing loss, also termed presbycusis.

Objective To investigate the effects of Bax on the cochlea in D-galactose-induced aging rats and the mech-anism of cell apoptosis in the peripheral auditory system in presbycusis. Methods Thirty-six 1-month male Sprague-Dawley rats were randomly divided into two groups (18 in each):①rats in D-galactose group received aily subcutaneous injection of D-gal at 500 mg/kg for 8 weeks, and②rats in the control group received the same volume of the vehicle (0.9%saline) once a day for 8 weeks. After the experiment termination, cochlear tissues were harvested. The accumulation of mitochondrial DNA (mtDNA) common deletion (CD) was detected by real-time PCR. Protein levels of Bax and Cleaved caspase-3 were evaluated by western blot and immunohistochemical analysis. Apoptotic cells were determined by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end-labelling (TUNEL) assay. All experimental data was analyzed using two-sample t-test. Results In comparison with the control group, the accumulation of mtDNA CD in the cochlea in rats of the D-galactose group was significantly increased (t=6.631, P<0.01). Protein expressions of Bax and Cleaved caspase-3 in the cochlea were also significantly increased (t=20.914 and 20.043, respectively, P<0.01). TUNEL-positive cells were found only in the cochlea of D-gal-induced aging rats, with a small number of these cells limited to the stria vascularis of the basal turn. Conclusions Bax-dependent cell apoptosis may contribute to D-galactose-induced aging process in the cochlea and may be an important reason for the occurrence of cell apoptosis in the peripheral auditory system in presbycusis.

Chronic administration of D-galactose (D-gal) is a useful method for establishing a model of natural aging in the auditory system. Previous studies have demonstrated that NADPH oxidases (NOXs) may be an important source of reactive oxygen species (ROS) in the peripheral auditory system (PAS) and cause an increase in mitochondrial DNA (mtDNA) common deletion (CD) levels in the PAS and central auditory system (CAS) of rats with D-gal-induced aging. However, the source of the ROS in the CAS and the mechanisms of age-related hearing loss (ARHL) have yet to be elucidated. In the present study, male Sprague Dawley rats were administered a daily injection of D-gal (150, 300 and 500 mg/kg, respectively) for eight weeks. All three doses of D-gal caused a significant increase in the expression of NOX2, 8-hydroxy-2-deoxyguanosine, a biomarker of DNA oxidative damage, and uncoupling protein 2, together with a decrease in the mitochondrial total antioxidant capabilities in the auditory cortex, as compared with the control rats (injected daily with the same volume of 0.9% saline for eight weeks). The levels of the mtDNA CD were also increased in the auditory cortex of the D-gal-induced aging rats. These findings suggest that both NOX- and mitochondria-associated ROS generation may contribute to mtDNA oxidative damage in the auditory cortex of the CAS of D-gal-induced aging rats. This study may provide novel insight into the development of ARHL.

目的 研究NADPH氧化酶(NADPH oxidase,NOX)在D-半乳糖诱导的老化大鼠外周听觉系统耳蜗和中枢听觉系统听皮层组织中的表达,探讨老年性耳聋氧化性损伤的发生机制.方法 48只1个月龄雄性Spragua-Dawley大鼠按数字随机法分成两组,每组各24只.D-半乳糖组:每日颈背部皮下注射D-半乳糖(500 mg/kg),连续8周;对照组:每日颈背部皮下注射同体积的生理盐水,连续8周.造模完成后,取两组大鼠外周听觉系统耳蜗和中枢听觉系统听皮层组织,利用免疫组织化学法检测DNA氧化损伤生物标记物8-羟基-2-脱氧鸟苷(8-hydroxy-2-deoxyguanosine,8-OHdG);利用实时定量PCR检测线粒体DNA(mitochondrial DNA,mtDNA)普遍缺失(common deletion,CD)和NOX基因的表达;利用Western blot检测NOX蛋白表达;应用Taqman PCR检测mt DNA CD.所有实验数据采用两样本的t检验进行分析.结果 D-半乳糖组和对照组大鼠相比较,D-半乳糖诱导的老化大鼠8-OHdG的表达在耳蜗和听皮层组织中明显增多,两组比较差异具有统计学意义(t分别为6.341、10.589,P均＜0.05);NOX3基因和蛋白的表达在耳蜗组织中明显增强,两组比较差异具有统计学意义(t分别为8.491、18.983,P均＜0.05);NOX2基因和蛋白的表达在听皮层组织中明显增强,两组比较差异具有统计学意义(t分别为3.548、8.219,P均＜0.05);mtDNA CD的累积在耳蜗和听皮层组织中明显增多,两组比较差异具有统计学意义(t分别为9.796、7.901,P均＜0.05).结论 在听觉系统老化过程中,NOX是活性氧的重要来源,可能是导致老年性耳聋氧化性损伤发生的重要原因.

Acanthus ilicifolius alkaloid A (4-hydroxy-2(3H)benzoxazolone, HBOA) is a naturally occurring compound that has been separated from Acanthus ilicifolius. Previous studies have reported the beneficial effects of HBOA on HSC-T6 cells. This study was undertaken in order to synthesize HBOA and two of its derivatives, specifically, 4-acetoxy-2(3H)-benzoxazolone (AcO-BOA) and 3-acetyl-4-acetoxy-2-benzoxazolone (TC-3), and to investigate the hepatoprotective potentials of these three compounds on CCl4-induced liver injury in mice. HBOA was prepared from 2-nitroresorcinol by a 'one pot' reduction and subsequent cyclization with urea. The acyl derivatives, AcO-BOA and TC-3, were prepared from HBOA using a substitution reaction. The compounds were synthesized with good yields (63.08-68.22%). An acute liver injury model was established by administering CCl4 intraperitoneally to Kunming mice. The mice were then intragastrically administered bifendate (150 mg/kg) or the synthesized compounds at three different doses (200, 100 and 50 mg/kg). The treatment with CCl4 was observed to increase the levels of aminotransferase (ALT), aspartate aminotransferase (AST), lactic dehydrogenase (LDH) and malondialdehyde (MDA) and decrease the levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione peroxidase (Gpx) in the liver tissues of the mice. Furthermore, treatment with CCl4 elevated the expression level of the proinflammatory mediator TNF-α. However, HBOA and its derivatives attenuated the changes induced by CCl4. Furthermore, CCl4-induced histopathological changes were reduced by treatment with these compounds. These results suggest that HBOA and its acyl derivatives are able to significantly alleviate the hepatotoxicity induced by CCl4 in mice.

Aim To explore the effects of Clausena-mide(Clau) on hippocampus cyclooxygenase-2 (COX-2) mRNA and protein expressions in diabetic rats. Methods The diabetic rat model was produced by injecting streptozotocin (STZ,48 mg·kg-1). After 3 months,the COX-2 gene and protein expressions in hippocampus of diabetic rats were detected by RT-PCR and immunohistochemistry respectively. Results ① The results of RT-PCR showed that the expression of COX-2 mRNA in hippocampus of diabetic group rats increased significantly (P0.01),however,the expression of COX-2 mRNA in the hippocampus of the rats in clau treatment groups (50,25 mg·kg-1) decreased significantly (P0.01).② The results of immunohistochemistry showed that the protein expression of COX-2 in hippocampus of diabetic group rats increased significantly (P0.01),but Clau could inhibit the expressions of COX-2(P0.01).Conclusion Clausenamide can significantly inhibit COX-2 mRNA and protein expressions in hippocampus,which may account for the protective effects.

Objective: To study the effect of nitidine chloride in inducing apoptosis of human hepatoma cell SMMC-7721. Methods: Cell viability was measured by MTT assay in human hepatoma cell line SMMC-7721. Morphological changes of cell nuclei were observed by Hoechst 33258 staining. Flow cytometry was performed to measure the cell cycle and cell apoptosis. Results: Proliferation of SMMC-7721 cells was significantly inhibited by nitidine chloride in concentration- and time- dependent manners in vitro, the 50% inhibition concentration (IC 50) value at 24, 48, and 72 h being (19.5±1.8), (3.8±0.2) and (2. 8 ±0.1) mg/L, respectively. The chromatine concentration and apoptotic cells with high concentrations were observed by Hoechst 33258 staining. The percentage of apoptotic cells increased in nitidine chloride -treated groups compared with that in the control group. The percentage of cells in G 2 /M phase increased and that of G 0/G 1, S phases was decreased according to flow cytometry determination. After treatment with nitidine chloride, apoptosis rate of SMMC-7721 cells increased with the increase of concentration. Conclusion: Nitidine chloride significantly inhibits the growth of SMMC-7721 cells. The anti-hepatoma mechanism of SMMC-7721 cells may be associated with the changes of cell cycle and inducing cell apoptosis.

目的:研究黄皮酰胺(Clausenmaide,Clau)对糖尿病导致的学习记忆障碍大鼠行为学和血糖的影响.方法:50只大鼠随机分为正常对照组、糖尿病模型组、胰岛素组、Clau高剂量组(50 mg/kg)和Clau低剂量组(25 mg/kg).腹腔注射链脲佐菌素建立糖尿病模型,以Morris水迷宫法考察各组大鼠的学习记忆能力,用血糖GOD-PAP试剂盒法检测血浆葡萄糖浓度.结果:3个月后,比较第7天大鼠在目标区域的时间,模型组大鼠为(42.86±8.69)s,Clau低剂量组大鼠为(37.56±11.48)s,与模型组相比明显缩短(P＜0.05);大鼠第一次穿越目标区域的时间模型组为(51.79±40.66)s,Clau低剂量组为(22.27±25.24)s,较模型组显著缩短(P＜0.05);给药后比较各组血糖值,模型组为(12.87±0.56)mmol/L,Clau高、低剂量组分别为(12.49±0.52)mmol/L、(12.38±0.44) mmol/L,与糖尿病模型组相比没有明显差异(P＞0.05).结论:Clau对糖尿病导致的学习记忆障碍大鼠在行为学上具有一定的保护作用,但并没有降血糖的作用.

Aim To explore the effects of clausenamide(Clau) on hippocampal heme oxygenase(HO-1 and HO-2) expressions in diabetic rats.Methods The diabetic rats model was produced by injecting streptozotocin (48 mg·kg-1). After 3 months,the HO-1 and HO-2 mRNA and protein expressions in hippocampus of diabetic rats were detected by RT-PCR and immunohistochemistry respectively.Results ① The results of RT-PCR showed that the protein expressions of HO-1 and HO-2 mRNA in hippocampus of diabetic group were higher than those of control group(P0.01).The expressions of HO-1 and HO-2 mRNA of Clau-treated group (50,25 mg·kg-1) were lower than those of diabetic group(P0.01).② The results of immunohistochemistry showed that the protein expressions of HO-1 and HO-2 in hippocampus of diabetic group were obviously increased(P0.01),but Clau could inhibit the expressions of HO-1 and HO-2.Conclusion Clau can significantly inhibit HO-1 and HO-2 expressions in hippocampus,which may account for the protective effects.