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

[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(NaAsO_(2))(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)NaAsO_(2)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。

[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(NaAsO_(2))were given 2.5,5.0,and 10.0 NaAsO_(2)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)NaAsO_(2)for 4 h and then 10 mL·kg^(−1)Rosa roxburghii Tratt juice;the Rosa roxburghii Tratt juice control group was given 10 mL·kg^(−1)Rosa 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)andγ-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(RTqPCR)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 NaAsO_(2),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 NaAsO_(2),andγ-GT in the high dose group of NaAsO_(2)were significantly increased(P<0.05).The levels of TBA andγ-GT raised with the increase of arsenic dose(Ptrend<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 NaAsO_(2)(P<0.05),and there was a dose-response relationship between dose of arsenic and SAM and between dose of arsenic and SAM/SAH(Ptrend<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)andγ-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 of Gnmt in the high dose group of NaAsO_(2),and Nnmt and Pemt in the low,medium,and high dose groups of NaAsO_(2)were significantly increased(P<0.05).The expression levels of these three genes increased in a dose-dependent manner with NaAsO_(2)treatment(Ptrend<0.05).Gnmt,Nnmt,and Pemt 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 NaAsO_(2),the hepatic sinusoidal dilation and inflammatory infiltration of liver cells in the Rosa roxburghii Tratt juice intervention group were alleviated;theγ-GT and TBA decreased significantly,and recovered to the level of the control group;the mRNA expression levels of Gnmt,Nnmt,and Pemt 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 of Gnmt,Nnmt,and Pemt 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.