刺梨汁对砷诱导的大鼠肝脏S-腺苷甲硫氨酸消耗及肝损伤的改善作用
Rosa roxburghii Tratt juice can improve S-adenosylmethionine consumption and liver damage induced by arsenic in rats
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摘要:
背景 砷可引起肝脏功能紊乱、脂肪变性、炎症、纤维化等不同程度肝损伤，但其机制尚不明确且缺乏有效治疗手段。
目的 从S-腺苷甲硫氨酸（SAM）代谢角度初步探讨刺梨汁对砷致大鼠肝损伤的干预作用及机制。
方法 36只Wistar大鼠按随机数字法分为6组，每组6只，雌雄各半。低、中、高砷剂量组分别给予2.5、5.0、10.0 mg·kg−1亚砷酸钠（NaAsO2）（以体重计，后同）灌胃，对照组给予10 mL·kg−1去离子水灌胃；刺梨汁干预组给予10 mg·kg−1 NaAsO2灌胃4 h后给予10 mL·kg−1刺梨汁灌胃；刺梨汁对照组给予10 mL·kg−1刺梨汁灌胃；每天灌胃1次，每周灌胃6 d，共处理4个月。苏木素-伊红染色（HE染色）观察大鼠肝脏组织病理学改变；酶循环法、速率法分别检测大鼠肝功能指标总胆汁酸（TBA）、谷氨酰胺转移酶（γ-GT）；超高效液相色谱（UPLC-MS）检测大鼠肝脏SAM、
S -腺苷同型半胱氨酸（SAH）含量；实时荧光定量逆转录PCR（RT-qPCR）检测大鼠肝脏中SAM代谢相关酶甘氨酸-N-甲基转移酶（GNMT）、烟酰胺N-甲基转移酶（NNMT）、磷脂酰乙醇胺N-甲基转移酶（PEMT）基因（Gnmt 、Nnmt 、Pemt ）的mRNA水平。结果 对照组大鼠肝细胞核染清晰，胞浆染色均匀，肝索呈放射状排列，无炎性细胞浸润；低、中、高砷剂量组可见不同程度的肝窦扩张、充血，炎性浸润。与对照组比较，中、高砷组TBA及高砷组γ-GT明显增加（
P <0.05），且随染砷剂量增加逐渐升高（P 趋势<0.05）。与对照组相比，低、中、高砷剂量组SAH含量明显增高，SAM含量、SAM/SAH明显降低（P <0.05），且均与染砷剂量呈剂量-反应关系（P 趋势<0.05）；SAM、SAM/SAH的降低与大鼠肝功能指标TBA（SAM：r =−0.569；SAM/SAH：r =−0.607）、γ-GT（SAM：r =−0.577；SAM/SAH：r =−0.622）的增加相关（P <0.05）。此外，与对照组相比，高砷剂量组Gnmt 及低、中、高砷剂量组Nnmt 、Pemt 基因mRNA表达水平均明显上升（P <0.05），且随染砷剂量升高呈上升趋势（P 趋势<0.05）。Gnmt 、Nnmt 、Pemt 基因mRNA表达与SAM（r =−0.490，r =−0.567，r =−0.593）、SAM/SAH（r =−0.433，r =−0.564，r =−0.746）均呈负相关（P <0.05）。与高砷剂量组相比，刺梨汁干预组大鼠肝细胞肝窦扩张减轻，炎性浸润情况得到缓解；γ-GT、TBA明显降低，且恢复至对照组水平；Gnmt 、Nnmt 、Pemt 基因mRNA表达明显降低，SAH含量下降，SAM含量及SAM/SAH回升（P <0.05）。结论 砷诱导的大鼠肝损伤与肝脏
Gnmt、Nnmt、Pemt 基因mRNA表达升高和SAM消耗密切相关。刺梨汁可能通过抑制砷诱导的Gnmt、Nnmt、Pemt 基因mRNA表达异常升高及SAM过度消耗改善砷引起的大鼠肝损伤。该研究结果为从维持SAM角度防治砷中毒肝损伤提供了依据。Abstract:Background Arsenic exposure can lead to liver dysfunction, liver steatosis, hepatitis, liver fibrosis, and other liver injuries, but its mechanism is still unclear and effective treatment methods are lacking.
Objective To investigate the potential intervention effect and associated mechanism of
Rosa roxburghii Tratt juice on arsenic-induced liver injury in rats from the perspective of S-adenosylmethionine (SAM) metabolism.Methods Thirty-six Wistar rats were randomly divided into six groups, six rats in each group, half male and half female. The low, medium, and high dose groups of sodium arsenite (NaAsO2) were given 2.5, 5.0, and 10.0 NaAsO2 mg·kg−1 (body weight, thereafter), respectively; the control group was given 10 mL·kg−1 deionized water; the
Rosa roxburghii Tratt juice intervention group was given 10 mg·kg−1 NaAsO2 for 4 h and then 10 mL·kg−1Rosa roxburghii Tratt juice; theRosa roxburghii Tratt juice control group was given 10 mL·kg−1Rosa roxburghii Tratt juice by gavage. All rats were gavaged once a day, 6 d per week for 4 months. Histopathological changes of rat liver were observed by hematoxylin-eosin (HE) staining. Total bile acids (TBA) and γ-glutamyl transpeptidase (γ-GT) were detected by enzyme cycle method and rate method, respectively. Ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS) was used to evaluate SAM and S-adenosylhomo cysteine (SAH) in rat liver. Real-time fluorescence quantitative reverse transcriptional PCR (RT-qPCR) was used to detect the mRNA expression levels of the genes of glycine-N-methyltransferase (Gnmt ), nicotinamide-N-methyltransferase (Nnmt ), and phosphatidylethano-lamine N-mathyltransferase (Pemt ).Results In the control group, the liver nuclei were stained clearly, the cytoplasm was stained uniformly, and the liver cords were arranged radially without inflammatory infiltration. In the low, medium, and high dose groups of NaAsO2, hepatic sinusoidal dilation, congestion, and inflammatory infiltration were observed to different degrees. Compared with the control group, TBA in the medium and high dose groups of NaAsO2, and γ-GT in the high dose group of NaAsO2 were significantly increased (
P <0.05). The levels of TBA and γ-GT raised with the increase of arsenic dose (P trend<0.05). Compared with the control group, SAH content was significantly increased, SAM content and SAM/SAH were significantly decreased in the low, medium, and high dose groups of NaAsO2 (P <0.05), and there was a dose-response relationship between dose of arsenic and SAM and between dose of arsenic and SAM/SAH (P trend<0.05). Decreases of SAM and SAM/SAH were significantly correlated with increase of liver function indexes TBA (SAM:r =−0.569; SAM/SAH:r =−0.607) and γ-GT (SAM:r =−0.577; SAM/SAH:r =−0.622) (P <0.05) respectively. In addition, compared with the control group, the mRNA expression levels ofGnmt in the high dose group of NaAsO2, andNnmt andPemt in the low, medium, and high dose groups of NaAsO2 were significantly increased (P <0.05). The expression levels of these three genes increased in a dose-dependent manner with NaAsO2 treatment (P trend<0.05).Gnmt ,Nnmt , andPemt mRNA expressions were negatively correlated with SAM (r =−0.490, −0.567, −0.593) and SAM/SAH (r =−0.433, −0.564, −0.746) respectively (P <0.05). Compared with the high dose group of NaAsO2, the hepatic sinusoidal dilation and inflammatory infiltration of liver cells in theRosa roxburghii Tratt juice intervention group were alleviated; the γ-GT and TBA decreased significantly, and recovered to the level of the control group; the mRNA expression levels ofGnmt ,Nnmt , andPemt and the SAH content were significantly decreased, and SAM content and SAM/SAH were increased (P <0.05).Conclusion Arsenic-induced liver injury in rats is closely related to increased expression of
Gnmt ,Nnmt ,Pemt and SAM consumption.Rosa roxburghii Tratt juice may ameliorate arsenic-induced liver injury in rats by inhibiting the abnormal mRNA expression ofGnmt ,Nnmt , andPemt as well as overconsumption of SAM. This study provides a basis for prevention and treatment of arsenic-induced liver injury from the perspective of SAM maintenance.
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