药学学报

	Day 1 and day 2	Day 3 and day 4	Day 5 and day 6	After day 7
MCS∶MCD	3∶1	2∶1	1∶1	All MCD

	Day 1 and day 2	Day 3 and day 4	Day 5 and day 6	After day 7
MCS∶MCD	3∶1	2∶1	1∶1	All MCD

Step	Temperature/℃	Time/s	Cycle
Initial denaturation	95	30	1
Denaturation	95	5	40
Annealing	60	10
Extension	72	15

Step	Temperature/℃	Time/s	Cycle
Initial denaturation	95	30	1
Denaturation	95	5	40
Annealing	60	10
Extension	72	15

Gene	Forward primer (5′→3′)	Reverse primer (5′→3′)
Il-6	AGTTGCCTTCTTGGGACTGA	TCCACGATTTCCCAGAGAAC
Il-1β	ATGAGAGCATCCAGCTTCAA	TGAAGGAAAAGAAGGTGCTC
Tnfα	CATCTTCTCAAAATTCGAGTGACAA	TGGGAGTAGACAAGGTACAACCC
Il-10	TGAATTCCCTGGGTGAGAAG	CTCTTCACCTGCTCCACTGC
Actin	GGCTGTATTCCCCTCCATCG	CCAGTTGGTAACAATGCCATGT

Gene	Forward primer (5′→3′)	Reverse primer (5′→3′)
Il-6	AGTTGCCTTCTTGGGACTGA	TCCACGATTTCCCAGAGAAC
Il-1β	ATGAGAGCATCCAGCTTCAA	TGAAGGAAAAGAAGGTGCTC
Tnfα	CATCTTCTCAAAATTCGAGTGACAA	TGGGAGTAGACAAGGTACAACCC
Il-10	TGAATTCCCTGGGTGAGAAG	CTCTTCACCTGCTCCACTGC
Actin	GGCTGTATTCCCCTCCATCG	CCAGTTGGTAACAATGCCATGT

大萼金丝桃素B抑制mTORC1信号通路改善非酒精性脂肪肝炎的作用和机制研究

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张艳秋 ^# , 何蒙蒙 ^# , 李学炎 , 徐文军 , 张浩 ^*

药学学报 | 研究论文 2023,58(8): 2391-2401

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药学学报 |研究论文 2023 , 58 (8) : 2391 -2401

大萼金丝桃素B抑制mTORC1信号通路改善非酒精性脂肪肝炎的作用和机制研究

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张艳秋^#, 何蒙蒙^#, 李学炎, 徐文军, 张浩^*

作者信息

中国药科大学中药学院, 天然药物活性组分与药效国家重点实验室, 江苏省天然活性物质发现与研究重点实验室, 江苏南京 211198

通讯作者:

*张浩, Tel: 86-25-83271402, E-mail: zhanghao@cpu.edu.cn

Hypercalin B alleviates nonalcoholic steatohepatitis progression via suppressing mTORC1 signaling pathway

Yan-qiu ZHANG, Meng-meng HE, Xue-yan LI, Wen-jun XU, Hao ZHANG^*

Affiliations

Jiangsu Key Laboratory of Bioactive Natural Product Research, State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, China

出版时间: 2023-08-12 doi: 10.16438/j.0513-4870.2023-0543

文章导航

摘要

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非酒精性脂肪肝炎(nonalcoholic steatohepatitis, NASH) 全球发病率持续攀升, 其发病机制复杂, 临床上尚无有效治疗手段。前期研究成果显示, RNA解旋酶DDX蛋白5 (DEAD box protein 5, DDX5) 可显著缓解小鼠NASH进程, 本研究对课题组天然产物库进行筛选, 发现中药黄花香(Hypericum beanii N. Robson) 活性成分大萼金丝桃素B (hypercalin B, HB) 可剂量依赖性上调DDX5蛋白表达量。本实验通过构建棕榈酸(palmitic acid, PA) 刺激的NASH体外模型以及胆碱-蛋氨酸缺乏饮食(methionine- and choline-deficient diet, MCD) 诱导的NASH动物模型, 采用不同浓度的HB处理, 考察HB缓解NASH进程的效果及作用机制。本文中所有动物实验都获得中国药科大学伦理学委员会批准(批准号: 2021-02-003)。体外模型结果显示, HB可显著减少游离脂肪酸(free fatty acid, FFA) 诱导的细胞内脂质沉积。动物实验结果显示, HB可显著减少NASH小鼠肝脏中脂质沉积; 降低血清谷草转氨酶(aspartate transaminase, AST) 和谷丙转氨酶(alanine transaminase, ALT) 水平, 改善肝损伤; 减少肝脏促炎因子白介素6 (interleukin 6, IL-6)、白介素1β (interleukin 1β, IL-1β) 和肿瘤坏死因子α (tumor necrosis factor α, TNFα) 等mRNA表达量, 抑制肝脏炎症激活。进一步研究表明, HB可减少雷帕霉素靶蛋白(mechanistic target of rapamycin, mTOR) 磷酸化水平, 降低脂肪酸合成关键蛋白固醇调节元件结合蛋白1 (sterol regulatory element-binding protein 1, SREBP1) 和脂肪酸合成酶(fatty acid synthase, FASN) 表达量, 从而改善脂代谢, 缓解NASH进程, 且HB该作用依赖于DDX5。综上, HB通过上调DDX5蛋白抑制雷帕霉素靶蛋白复合物1 (mechanistic target of rapamycin complex 1, mTORC1) 信号通路激活, 改善脂代谢紊乱、抑制炎症, 在体内外表现出良好的抗NASH活性。

关键词

非酒精性脂肪肝炎 / 大萼金丝桃素B / 雷帕霉素靶蛋白复合物1 / 脂代谢 / 炎症

Abstract

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The global incidence rate of nonalcoholic steatohepatitis (NASH) continues to rise. The pathogenesis of NASH is complex, and there is no effective clinical treatment. Previous study has shown that DEAD box protein 5 (DDX5) can significantly alleviate the NASH process in mice. This study screened the natural product library of the research group and found that the active compound hypercalin B (HB) in Hypericum beanii N. Robson, a traditional Chinese medicine, can upregulate the expression of DDX5 protein in a dose-dependent manner. In this study, an in vitro model of NASH stimulated by palmitic acid (PA) and an animal model of NASH induced by the methionine- and choline-deficient diet (MCD) were constructed. Different concentrations of HB were used to investigate the effect and mechanism of HB in alleviating NASH progression. All animal experiments in this paper were approved by the Ethics Committee of China Pharmaceutical University (NO: 2021-02-003). In vitro model results showed that HB significantly reduced the intracellular lipid deposition induced by free fatty acid (FFA). Animal experiments showed that HB improved liver injury by significantly reducing lipid accumulation in the liver of NASH mice, and reducing serum aspartate transaminase (AST) and alanine transaminase (ALT) levels. Moreover, HB could inhibit liver inflammation by reducing the mRNA levels of liver pro-inflammatory cytokines including interleukin 6 (IL-6), interleukin 1β (IL-1β), and tumor necrosis factor α (TNFα). Further research showed that HB could reduce the phosphorylation level of the mechanical target of rapamycin (mTOR) and reduce the expression of sterol regulatory element binding protein 1 (SREBP1) and fatty acid synthase (FASN), thereby improving lipid metabolism and alleviating NASH progression, and the effects of HB against NASH were dependent on DDX5. In conclusion, HB can improve lipid metabolism and inhibit inflammatory activation by suppressing mTORC1 pathway via upregulating DDX5 protein, and showed promising anti-NASH activity in vitro and in vivo.

Key words

nonalcoholic steatohepatitis / hypercalin B / mechanistic target of rapamycin complex 1 / lipid metabolism / inflammation

引用本文

张艳秋, 何蒙蒙, 李学炎, 徐文军, 张浩. 大萼金丝桃素B抑制mTORC1信号通路改善非酒精性脂肪肝炎的作用和机制研究. 药学学报, 2023 , 58 (8) : 2391 -2401 . DOI: 10.16438/j.0513-4870.2023-0543

Yan-qiu ZHANG, Meng-meng HE, Xue-yan LI, Wen-jun XU, Hao ZHANG. Hypercalin B alleviates nonalcoholic steatohepatitis progression via suppressing mTORC1 signaling pathway[J]. Acta Pharmaceutica Sinica, 2023 , 58 (8) : 2391 -2401 . DOI: 10.16438/j.0513-4870.2023-0543

正文

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非酒精性脂肪肝病(nonalcoholic fatty liver disease, NAFLD) 是指无大量饮酒和其他肝损伤因素所引起的、以肝脏中甘油三酯(triglyceride, TG) 积累增多为特征的疾病, 包括非酒精性单纯性脂肪肝、非酒精性脂肪肝炎(nonalcoholic steatohepatitis, NASH)、肝纤维化、肝硬化和肝癌。其中, NASH以肝细胞大泡性脂肪变、小叶炎症和气球样变为特征, 并伴有不同程度纤维化, 可发展为肝硬化甚至肝癌^[1]。我国超过2亿人患有NAFLD, 其中20%~60%是NASH^[2], 且NASH已逐渐成为导致肝癌发生的最主要原因之一^[3]。早期, 人们提出“二次打击”理论解释NASH的发病机制, 但这种理论过于简单, 无法概括NASH的复杂性。由于多种平行因素与NASH的发生发展有关, 现如今, “多重打击”假说被越来越多的人们接受, 即由遗传易感性、肥胖、脂代谢、先天性免疫、炎症、自噬、内质网应激等多种因素导致NASH产生^[4]。目前, 饮食控制和锻炼减重等方法是NASH/NAFLD治疗的基石, 但很多患者不易坚持; 此外有分析表明, 全球NAFLD人口中大约40%为非肥胖人群, 减重疗法并不完全适用^[5]。目前, 临床上尚无有效控制NASH及其进展的药物, 医疗需求远未得到满足。

雷帕霉素靶蛋白复合物1 (mechanistic target of rapamycin complex 1, mTORC1) 信号通路在细胞中感应内外营养素、激素和能量变化, 调节细胞增殖、蛋白合成和自噬等生命活动上具有重要作用^[6]。脂代谢紊乱是NASH的一个重要表现形式和诱发因素, 而mTORC1是调控脂代谢的关键通路。mTORC1通过调控脂肪酸合成关键蛋白固醇调节元件结合蛋白1 (sterol regulatory element-binding protein 1, SREBP1) 促进从头脂肪生成(de novo lipogenesis, DNL), 从而导致脂代谢紊乱、产生脂毒性^[7]。脂毒性一方面可导致肝脏脂肪变, 另一面可激活炎性因子如肿瘤坏死因子α (tumor necrosis factor α, TNFα) 和白介素1β (interleukin 1β, IL-1β) 等, 加剧肝脏炎症反应, 促进NASH进程^[8]。

中药及天然产物在NASH治疗中具有疗效显著、毒副作用小等明显优势。本课题组前期发现, RNA解旋酶DDX蛋白5 (DEAD box protein 5, DDX5) 可通过抑制mTORC1信号通路激活缓解NASH发生发展^[9], 基于此, 本课题组对实验室天然活性产物库进行了筛选, 发现大萼金丝桃素B (hypercalin B, HB) 能够剂量依赖性地提高DDX5表达量。HB由栽秧花根中分离得到, 有报道其具有一定的抗肿瘤作用^[10]。栽秧花(Hypericum beanii N. Robson) 为藤黄科金丝桃属植物, 又名黄花香, 作为传统中药, 黄花香主要用于治疗肝炎、湿热黄疸、淋病、泄泻、痢疾、黄水疮等^{[11, 12]}。为研究HB对NASH进程的影响, 本研究通过构建棕榈酸(palmitic acid, PA) 刺激的细胞模型以及胆碱和蛋氨酸缺乏饮食(methionine- and choline-deficient diet, MCD) 诱导的NASH小鼠模型, 在体内外考察HB的抗NASH作用及机制, 发现HB可显著抑制mTORC1信号通路激活并缓解小鼠NASH进程。本研究可为金丝桃属类化合物作为一种有前景的抗NASH先导化合物提供理论和实验依据。

材料与方法

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实验动物 雄性C57BL/6J小鼠(6~8周龄), 购买于北京维通利华实验动物技术有限公司南京分公司[SPF级, 合格证号: SCXK (苏) 2016-0003], 饲养于中国药科大学药学动物实验中心。所有动物实验都获得中国药科大学伦理学委员会批准(批准号: 2021-02-003)。实验动物在12 h明暗交替的SPF级动物房中饲养, 自由进食饮水, 预适应环境7天。

药物与实验试剂 HB来源于本实验室, 从栽秧花根中分离得到: 栽秧花干燥根切片后95%乙醇浸提3次, 回收溶剂至干后加水混悬, 加石油醚萃取3次。石油醚部位浓缩, 浸膏经硅胶柱色谱, 用石油醚/乙酸乙酯梯度洗脱, 收集纯乙酸乙酯段回收溶剂得浸膏。再经过中压制备柱色谱, 在73%甲醇/水洗脱条件时得到HB纯品。MCD (南通特洛菲饲料科技有限公司, 货号TP3005); 血清谷草转氨酶(aspartate transaminase, AST)、谷丙转氨酶(alanine transaminase, ALT) 测试盒(南京建成生物工程研究所, 货号分别为C010-2-1、C009-2-1); ChamQ SYBR qPCR Master Mix、HiScript Ⅱ Q RT SuperMix for qPCR (南京诺唯赞生物科技股份有限公司, 货号分别为Q321-02、R222-01); Total RNA提取试剂盒(上海奕杉生物科技有限公司, 货号ES-RN001); DDX5抗体(货号9877)、脂肪酸合成酶(fatty acid synthase, FASN) 抗体(货号3180)、雷帕霉素靶蛋白(mechanistic target of rapamycin, mTOR) 抗体(货号2983)、p-mTOR (Ser2448) 抗体(货号5536)、S6K抗体(货号2708)、p-S6K (Thr389) 抗体(货号9208)、cell lysis buffer (货号9803) (Cell Signaling Technology公司); 固醇调节元件结合蛋白1 (sterol regulatory element-binding protein 1, SREBP1) 抗体(Santa Cruz Biotechnology公司, 货号sc-13551); Actin抗体(上海翊圣生物科技有限公司, 货号30101ES60); PVDF膜(Bio-Rad公司, 货号1620177); EasySee Western blot kit (北京全式金生物技术股份有限公司, 货号DW101-01)。

PA诱导的细胞模型构建 配制20%无脂肪酸BSA (d-BSA) 溶液, 溶剂为PBS; 配制20 mmol·L^-1 PA溶液, 溶剂为0.1 mol·L^-1 NaOH溶液, 在70~75 ℃水浴锅中溶解配制, 充分皂化; 取等体积的d-BSA溶液迅速加入保温的PA溶液中, 可置于50~55 ℃水浴锅中助溶, 得到澄清的10 mmol·L^-1 PA + 10% d-BSA溶液; 将上述得到的溶液于超净台中过滤除菌, 即为棕榈酸脂性培养基(PA储存液), 分装后于4 ℃保存; 对照组配制: 20% d-BSA溶液混合等体积的0.1 mol·L^-1 NaOH溶液, 过滤除菌后分装保存; 取PA储存液及相应的对照溶液, 用细胞培养基稀释成相应终浓度, 培养16 h后收细胞或进行其他实验操作。

FFA诱导的细胞模型构建 配制20% d-BSA溶液, 溶剂为PBS; 配制20 mmol·L^-1 FFA [PA∶OA (oleic acid, 油酸) = 1∶2] 溶液, 溶剂为0.1 mol·L^-1 NaOH溶液, 在70~75 ℃水浴锅中溶解配制, 充分皂化; 其余步骤同“PA诱导的细胞模型构建”。

油红O染色 将细胞接种至合适的孔板, FFA处理后多聚甲醛固定20 min, 60%异丙醇漂洗1 min, 油红O染色液浸染20 min, 60%异丙醇漂洗20 s至间质清晰, 水洗3次后加入蒸馏水覆盖细胞, 采用倒置显微镜采集图像。

MCD模型构建 采用40只6~8周龄的C57BL/6J雄鼠, 随机分为5组, 分别为对照饲料组MCS (methionine and choline-sufficient diet)、模型组MCD、MCD + HB 10 mg·kg^-1、MCD + HB 20 mg·kg^-1、MCD + HB 40 mg·kg^-1, 每组8只, 第1周过渡喂养, 第3周开始灌胃给予HB, 4周后, 取小鼠全血及肝脏进行后续分析(表 1)。

血清生化指标检测 全血室温静置2 h, 4 ℃、4 000 r·min^-1离心10 min后上清即为血清, 血清ALT和AST根据说明书按照标准操作程序测定。

肝脏病理切片 取小鼠肝脏固定于4%多聚甲醛24 h以上, 石蜡包埋后切片, 按照说明书进行HE、油红O、Masson和Sirius Red染色, 显微镜观察并采集图像分析。

实时荧光定量PCR (real time quantitative polymerase chain reaction, RT-qPCR) 首先采用RNA提取试剂盒提取小鼠肝脏中总RNA, Nanodrop测定RNA浓度, 取1 µg RNA逆转录成cDNA, 于-20 ℃保存作后续实验用。将适量的cDNA用RNase free dH₂O稀释5倍, 按说明书配置好PCR反应液, 反应条件和引物序列如表 2、3所示。

蛋白免疫印迹(Western blot, WB) 在液氮速冻后的小鼠肝脏或冰上收集的细胞中加入含有蛋白酶抑制剂的裂解液, 超声破碎, 4 ℃、12 000 r·min^-1离心10 min, 取上清液进行蛋白定量, 加入2×上样缓冲液, 沸水煮10 min后进行SDS聚丙烯酰胺凝胶电泳, 转印至PVDF膜, 5% BSA室温封闭1 h, 一抗4 ℃孵育过夜, 二抗室温孵育2 h, 采用ECL显影液于Bio-Rad ChemiDocXRS+成像系统中进行分析。

统计学方法 采用GraphPad Prism 8软件作统计分析, 数据用平均值±标准误(mean ± SEM) 表示, 两组数据采用t检验统计, 多组数据采用单因素方差分析(one-way ANOVA) 统计, 分析结果^*表示P < 0.05, 有显著性差异; ^**表示P < 0.01, 有特别显著性差异; ^***表示P < 0.001, 有极显著性差异。

结果

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1 HB显著减少细胞内脂质沉积

在PA刺激的HepG2细胞模型中给予不同剂量的HB (图 1A), 发现12.5 μmol·L^-1以下时不存在细胞毒(图 1B), 进一步采用WB检测DDX5蛋白表达量, 发现HB可剂量依赖性地上调DDX5蛋白表达量(图 1C)。进一步, 在FFA处理的L02细胞中给予HB, 采用油红O染色显示细胞中脂滴大小和数量, 实验结果显示, 随着FFA浓度的提高, 细胞中脂质沉积明显加重, 而给予HB后, 则能明显减少细胞脂滴大小和数量(图 1D)。在HepG2细胞中, 得到同样结果(图 1E)。以上结果表明, HB可显著减少NASH细胞模型中脂质沉积。

2 HB改善NASH小鼠肝损伤和脂质沉积

首先考察HB的体内毒性, HE染色结果显示, 高剂量下HB对小鼠各脏器无明显毒性(图 2A)。进一步, 采用MCD构建NASH小鼠模型, 灌胃给予不同剂量的HB, 小鼠肝脏HE染色结果显示, 模型组小鼠肝损伤严重, 给予HB后, 随着HB剂量的增加, 小鼠肝损伤程度逐渐减轻; 油红O染色结果显示, HB能够明显减少小鼠肝脏中脂质沉积, 且具有剂量依赖性(图 2B)。血清AST和ALT结果同样表明, HB能够明显缓解NASH小鼠肝损伤(图 2C、D)。以上结果表明, HB能够明显缓解MCD诱导的NASH小鼠模型肝损伤及脂质沉积。

3 HB改善NASH小鼠肝纤维化及炎症反应

NASH发病机制复杂, 其核心除脂质沉积外, 纤维化和炎症激活同样是驱动NASH进程的重要因素。通过对小鼠肝组织切片进行Masson和Sirius red染色发现, 对照组中几乎没有胶原纤维沉积, 模型组中胶原沉积较多, 给予HB后胶原染色面积明显减少, 且高剂量组的改善效果更为明显(图 3A), 提示HB能够减轻NASH小鼠肝脏纤维化程度。通过检测小鼠肝脏中炎症相关基因发现, 模型组小鼠肝脏中促炎因子Il-6、Il-1β和Tnfα的mRNA表达量明显升高, 而抗炎因子Il-10 mRNA表达量明显下降, 给予HB后则能够明显减少促炎因子Il-6、Il-1β和Tnfα表达量, 显著提高抗炎因子Il-10表达量, 并具有剂量依赖性, 提示HB能够缓解NASH小鼠肝脏炎症激活。以上结果表明, HB通过减轻肝脏纤维化程度及抑制肝脏炎症激活, 从而缓解小鼠NASH进程。

4 HB通过减少小鼠肝脏中脂肪酸合成减轻脂质沉积

脂代谢紊乱是NASH的一个重要表现形式和诱发因素, 其中脂肪酸合成关键基因SREBP1和FASN通过促进DNL干扰脂代谢从而促进肝脏脂肪变^[13]。本研究通过检测小鼠肝脏相关蛋白表达量, 发现随着HB剂量的提高, 虽然不影响SREBP1前体(pSREBP1) 表达量, 但能明显减少成熟型SREBP1 (mSREBP1) 的表达量, 同时显著降低FASN表达量(图 4A、B), 表明HB可通过抑制脂肪酸合成从而改善NASH小鼠肝脏脂代谢, 减轻小鼠肝脏脂质沉积。另一方面, 在PA刺激的HepG2细胞中, HB同样可以减少mSREBP1与FASN蛋白表达量, 且具有剂量依赖性(图 4C、D)。

5 HB通过抑制mTORC1信号通路激活改善NASH进程

前期通过筛选, 发现在PA刺激的细胞模型中, HB能够剂量依赖性地上调DDX5蛋白表达量, 在MCD诱导的NASH小鼠模型中给予HB处理后, 随着剂量的增加, 小鼠肝脏中DDX5表达量随之升高(图 5A、B)。进一步研究发现, HB处理后不影响mTOR总蛋白表达量, 但可剂量依赖性抑制小鼠肝脏中p-mTOR表达水平(图 5A、B), 表明HB可抑制小鼠肝脏中mTORC1信号通路激活; 在PA刺激的HepG2细胞模型中, HB不影响mTOR和S6K总蛋白表达量, 但可剂量依赖性降低p-mTOR和p-S6K表达量(图 5C、D), 表明HB可抑制PA诱导的NASH细胞模型中mTORC1信号通路激活。以上结果提示, HB通过抑制mTORC1信号通路激活从而缓解NASH发生发展。

进一步, 在HepG2细胞中下敲DDX5后再给予HB, 检测mTORC1信号通路和脂肪酸合成相关蛋白表达量, 发现下敲DDX5后HB对mTORC1信号通路及FASN、SREBP1的调控作用被逆转, 说明HB改善NASH进程依赖于DDX5 (图 5E、F)。

讨论

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NAFLD已成为最常见的慢性肝病, 其中NASH是NAFLD进展期, 可发展为肝纤维化、肝硬化甚至肝癌, 目前尚无FDA正式批准治疗NASH的药物。NASH的发病机制较为复杂, 经历了“双重打击”到“多重打击”学说的转变, 其核心是脂质沉积、炎症和纤维化^[14]。

MCD模型是用于诱导小鼠NASH模型最常用的饮食模型之一, 已使用40余年, 其含有40%果糖和10%脂肪, 但缺乏蛋氨酸和胆碱, 导致脂肪酸摄入增加、极低密度脂蛋白(very low density lipoprotein, VLDL) 分泌减少, 进而导致肝内脂质蓄积, 可在最短的时间内产生最严重的NASH表型。小鼠喂食MCD饮食体重减轻, 随着白色脂肪组织的减少, 肝脏也会缩小^[15-17]。2~4周时, 肝脏即发生大泡性脂肪变, 此后巨噬细胞活化浸润肝脏、核转录因子κB (nuclear factor kappa B, NFκB) 激活, 伴随着促炎因子IL-6、转化生长因子β (transforming growth factor β, TGFβ) 和TNFα的增加, 小鼠肝脏炎症反应增加, 并可诱导肝脏纤维化^[18-20]。本研究发现, HB可缓解MCD诱导的小鼠肝脏脂质蓄积和炎症激活。

在NASH发展前期, 细胞中TG大量累积从而导致脂代谢紊乱, 除了FFA形成的TG, DNL也是TG的一个重要来源, DNL在NASH发生发展过程中具有重要作用, 占人类受试者肝脏TG的26%^[21]。在DNL过程中, 葡萄糖首先转化为乙酰辅酶A, 乙酰辅酶A通过乙酰辅酶A羧化酶形成丙二酰辅酶A, 脂肪酸合成限速酶FASN则催化乙酰辅酶A和丙二酰辅酶A合成PA, PA经硬脂酰辅酶A去饱和酶(stearoyl-CoA desaturase, SCD) 形成单不饱和脂肪酸, 继而通过甘油-3-磷酸酰基转移酶(glycerol-3-phosphate acyltransferase, GPAT) 催化TG合成的初始步骤, 最终经二酰基甘油酰基转移酶(diacylglycerol acyltransferase, DGAT) 催化合成TG^{[22, 23]}。在DNL调节过程中, 核转录因子SREBP1具有重要作用, SREBP1通过促进脂肪酸合成关键基因FASN等从而促进DNL, 加剧脂代谢紊乱、形成脂肪变, 最终促进NASH进展^{[24, 25]}。

mTORC1作为调节细胞代谢的关键信号通路, 主要通过增加合成代谢和抑制分解代谢过程来调控细胞生长等生命活动^[26]。mTORC1慢性激活可导致肥胖、肝脂肪变、胰岛素抵抗和纤维化等^[27]。mTORC1抑制剂被认为是潜在的NASH和HCC治疗方法。然而, 肝细胞特异性缺失mTORC1活性可导致肝损伤和炎症, 并显著提高肝癌发生率, 因此, 直接靶向mTORC1可能并不是NASH或相关肝癌治疗的有效策略^[26]。在NASH发生发展过程中, 脂代谢紊乱具有重要作用, 而mTORC1是影响脂代谢过程的重要信号通路。mTORC1信号通路过度激活导致下游S6K磷酸化水平升高, 而S6K可调节SREBP1c活性从而促进DNL, 干扰脂代谢促进肝脏脂肪变^[28]。除了异常增加的DNL, 脂肪酸氧化不足也是另一个促进NASH进展的重要因素, 肝脏自噬水平对于维持脂肪酸氧化平衡具有重要作用^[29-32]。肝脏中脂肪酸主要以脂滴的形式贮存, 脂滴主要通过自噬以脂噬的形式降解, 因此, 提高脂噬水平能够促进脂肪酸氧化, 降低脂毒性, 而自噬缺陷则会导致脂滴异常蓄积, 脂毒性增加, 继而加重NASH发展^[33-36]。作为自噬的直接上游调控中心, mTOR通过直接磷酸化ULK1抑制自噬小体形成从而降低自噬水平, 抑制脂噬发生, 最终促进NASH发展^{[37, 38]}。另一方面, 炎症激活也是加重NASH进程的重要因素, 其中对NLRP3炎症小体的研究最为广泛^[39-41], 脂质过度积累可持续激活NLRP3, 导致肝细胞损伤、细胞内容物(如ATP等) 释放, ATP又继续激活肝细胞和Kupffer细胞中NLRP3炎症小体, 形成恶性循环, 加重NASH进程^{[42, 43]}。NLRP3炎症小体是一种细胞内多蛋白复合物, 由NLRP3、ACS和caspase-1组成, 它的激活使caspase-1将促炎因子IL-1β和IL-18剪切为成熟形式释放出胞外, 继而引发一系列炎症反应。在NASH患者和动物模型的肝脏中, NLRP3和促炎因子IL-1β的表达量显著升高^[44]。有研究报道, mTORC1依赖的糖酵解能够促进NLRP3炎症小体激活^[45], 并且NLRP3和mTOR可直接相互作用调控炎症反应^[46], 这些都表明mTORC1与炎症发生之间存在密切联系。

在本课题组前期研究成果中, 发现DDX5蛋白通过抑制脂肪酸合成关键基因SREBP1、FASN和SCD, 抑制TG合成关键基因GPAT、DGAT, 以及提高肝脏自噬水平从而改善肝脏脂代谢, 进一步研究发现DDX5通过招募TSC1/2复合物至mTOR, 从而抑制肝脏mTORC1信号通路激活, 改善脂代谢紊乱、增加自噬活性、抑制NLRP3炎症小体激活, 进而缓解NASH发生发展^[9]。本研究前期通过筛选, 发现HB可剂量依赖性上调NASH小鼠和细胞模型中DDX5表达量, 并可抑制mTORC1信号通路激活; 在细胞中下敲DDX5后再给予HB, 发现HB失去了对mTORC1信号通路和脂肪酸合成相关蛋白的调控作用, 提示HB通过上调DDX5从而抑制mTORC1信号通路激活, 进而改善NASH进程。

综上所述, HB通过上调DDX5蛋白表达量抑制mTORC1信号通路激活, 从而抑制脂肪酸合成途径关键蛋白表达, 改善脂代谢, 并可减轻炎症反应, 在体内外水平表现出良好的抗NASH作用。本研究为中药黄花香在临床中治疗NASH提供了理论和实验依据。

作者贡献: 张艳秋和何蒙蒙负责细胞和动物实验、数据分析及文章撰写; 李学炎负责HB的提取分离和制备; 徐文军参与课题设计和指导; 张浩负责课题设计、指导和论文审阅。

利益冲突: 所有作者均声明不存在利益冲突。

基金

收起

国家自然科学基金资助项目(81872889)
国家自然科学基金资助项目(82074068)
江苏省自然科学基金资助项目(BK20221052)

参考文献

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2023年第58卷第8期

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doi: 10.16438/j.0513-4870.2023-0543

接收时间：2023-04-28
首发时间：2025-11-21
出版时间：2023-08-12

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收稿日期：2023-04-28
修回日期：2023-06-30

基金

国家自然科学基金资助项目(81872889)

国家自然科学基金资助项目(82074068)

江苏省自然科学基金资助项目(BK20221052)

作者信息

中国药科大学中药学院, 天然药物活性组分与药效国家重点实验室, 江苏省天然活性物质发现与研究重点实验室, 江苏南京 211198

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*张浩, Tel: 86-25-83271402, E-mail: zhanghao@cpu.edu.cn

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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