痛泻要方通过调控SREBP-1/SCD1通路改善5-HT诱导的BRL 3A细胞氧化应激损伤

doi:10.12122/j.issn.1673-4254.2026.06.018

摘要/Abstract

摘要：

目的研究SREBP-1/SCD1通路在5-HT致肝细胞BRL 3A细胞损伤中的作用，探讨痛泻要方含药血清对氧化应激损伤的保护作用及机制。方法制备含药血清及培养BRL 3A细胞：14只SD雄性大鼠予痛泻要方灌胃，每日1次，连续7 d，腹主动脉取血制备含药血清；用10%胎牛血清37 ℃、5%CO₂饱和湿度条件下培养。CCK-8法检测不同浓度5-HT（0.2、0.5、1、1.5 mmol/L）处理 BRL 3A 细胞48、72、96 h后细胞存活率。细胞培养给药后进行超氧化物歧化酶（SOD）、丙二醛（MDA）含量检测，流式法检测细胞活性氧（ROS）含量。采用JC-1染色检测细胞线粒体膜电位变化，细胞电镜观察线粒体形态变化。免疫荧光法检测细胞自噬小体（由MDC特异性标记）及其标志蛋白LC3B，荧光显微镜下观察自噬体及其标志蛋白在细胞内的位置、表达情况及细胞核的形态变化。Western blotting法检测细胞Cyt-c、Caspase3、Gsk3β、beclin-1、LC3Ⅰ/Ⅱ、p62，SREBP-1、SCD1，5-HT_2AR蛋白表达水平。结果通过CCK-8测定细胞最适宜的生长浓度为1.5 mmol/L（P<0.01）；与model组相比，TXYF组ROS含量显著降低（P<0.0001）、MDA含量降低（P<0.05）、SOD含量升高（P<0.01）。与model组相比，TXYF组线粒体膜电位降低，UR降低（P<0.001），LR升高（P<0.001）；在透射电子显微镜下可观察到TXYF组线粒体肿胀程度较model组明显减轻，大部分线粒体嵴结构形态恢复。与model组相比，TXYF组LC3B和MDC免疫荧光强度下降（P<0.05），自噬相关蛋白Beclin-1降低（P<0.05）、LC3Ⅱ/LC3Ⅰ降低、p62升高（P<0.01）；通过检测线粒体途径凋亡，TXYF组较model组Cyt-c降低（P<0.01），Caspase3及Gsk3β蛋白量降低但无统计学意义。与model组相比，TXYF组SREBP-1蛋白表达升高（P<0.05）、SCD1蛋白表达降低（P<0.01），5-HT_2AR蛋白表达降低（P<0.05）。结论痛泻要方含药血清可能通过SREBP-1/SCD1通路，调节SOD活力与自噬功能来减轻氧化应激和脂质过氧化损伤，对5-HT诱导的BRL 3A细胞损伤具有保护调节作用。

关键词: 痛泻要方, 5-羟色胺, BRL 3A细胞, 氧化应激, 细胞自噬

Abstract:

Objective To investigate the role of the SREBP-1/SCD1 pathway in mediating the protective effect of Tongxie Yaofang (TXYF) against 5-hydroxytryptamine (5-HT)-induced injury in BRL 3A cells. Methods Fourteen SD rats were gavaged with TXYF for 7 consecutive days to prepare TXYF-medicated serum. BRL 3A cells in routine culture were examined for changes in cell viability using CCK-8 assay following treatment with different concentrations of 5-HT. In BRL 3A cells treated with 5-HT and different concentrations of the medicated serum, SOD, MDA, and ROS levels were measured, and mitochondrial membrane potential and cell morphology were evaluated using JC-1 staining and electron microscopy; autophagosomes and LC3B expression were detected with immunofluorescence staining, and the protein expression levels of Cyt-c, caspase-3, Gsk3β, Beclin-1, LC3 I/II, p62, SREBP-1, SCD1, and 5-HT_2AR were analyzed using Western blotting. Results The optimal 5-HT concentration for modeling was 1.5 mmol/L. In 5-HT-treated cells, treatment with TXYF-medicated serum significantly reduced cellular ROS and MDA levels, increased SOD activities, lowered mitochondrial membrane potential, and alleviated mitochondrial swelling. The cells treated with TXYF-medicated serum also showed decreased expression levels of LC3B, MDC, Beclin-1, LC3 II/LC3 I, Cyt-c, SCD1 and 5-HT_2AR and increased expressions of p62 and SREBP-1 without significant changes in caspase-3 and Gsk3β expressions. Conclusion TXYF-medicated serum alleviate oxidative stress and autophagy dysfunction via the SREBP-1/SCD1 pathway to protect BRL 3A cells against 5-HT-induced injury.

Key words: Tongxie Yaofang, 5-hydroxytryptamine, BRL 3A cells, oxidative stress, autophagy

宋明朗, 苏海, 夏洪兴, 陈诗雨, 邵丽丽, 蒋志滨. 痛泻要方通过调控SREBP-1/SCD1通路改善5-HT诱导的BRL 3A细胞氧化应激损伤[J]. 南方医科大学学报, 2026, 46(6): 1385-1394.

Minglang SONG, Hai SU, Hongxing XIA, Shiyu CHEN, Lili SHAO, Zhibin JIANG. Mechanism of Tongxie Yaofang for reducing 5-HT-induced oxidative stress injury in BRL 3A cells: the mediating role of the SREBP-1/CD1 pathway[J]. Journal of Southern Medical University, 2026, 46(6): 1385-1394.

图/表 6

图1 不同浓度的5-HT培养基对BRL 3A细胞存活率的影响

Fig.1 Effects of different concentrations of 5-HT on survival rate of BRL 3A cells (Mean±SD, n=3). *P<0.05, **P<0.01 vs control group.

图2 痛泻要方对BRL 3A细胞氧化应激的影响

Fig.2 Effect of TXYF-medicated serum on oxidative stress in 5-HT-induced BRL 3A cells (Mean±SD, n=3). A: ROS expression level. B: MDA content. C: SOD activity. D: Flow cytometric analysis of ROS level. ***P<0.001, ****P<0.0001 vs control group; #P<0.05, ##P<0.01, ####P<0.0001 vs model group.

图3 痛泻要方对BRL 3A细胞线粒体功能的影响

Fig.3 Effect of TXYF-medicated serum on mitochondrial function in 5-HT-induced BRL 3A cells (Mean±SD, n=3). A: Flow cytometry for detecting mitochondrial membrane potential in each group. B: Percentages of UR in each group. C: Percentages of LR in each group. D: Observation of mitochondrial morphologies in each group using transmission electron microscopy. The black arrow indicates normal structure of mitochondria, and the red arrow indicates structural change in the mitochondria; the green and the blue arrow indicated structural changes of mitochondria after treatment with the antagonist and TXYF, respectively (Original magnification: ×12 000). ****P<0.0001 vs control group; ###P<0.001 vs model group.

图4 痛泻要方对BRL 3A细胞自噬能力的影响

Fig.4 Effect of TXYF-medicated serum on autophagy of 5-HT-induuyced BRL 3A cells (Mean±SD, n=3). A: LC3B confocal microscopy (×600). B: MDC confocal microscopy (×600). C: LC3B fluorescence intensity. D: MDC fluorescence intensity. E: Beclin-1 protein expression level. F: LC3Ⅰ protein expression level. G: LC3Ⅱ protein expression level. H: LC3Ⅱ/LC3Ⅰ protein expression level. I: p62 protein expression level. J: beclin-1, LC3Ⅰ, LC3Ⅱ, p62, GAPDH protein bands. **P<0.01, ***P<0.001, ****P<0.0001 vs control group; #P<0.05, ##P<0.01, ###P<0.001 vs model group.

图5 痛泻要方对5-HT刺激后的细胞凋亡相关蛋白表达的影响

Fig.5 Effect of TXYF-medicated serum on expressions of apoptosis-related proteins after 5-HT stimulation （Mean±SD, n=3）. A: Cyt-c intensity. B: Caspase-3 32 kD intensity. C: Caspase3-17 kDa intensity. D: GSK3β intensity. E: Cyt-c, caspase-3, GSK3β, and GAPDH protein bands.*P<0.05, ****P<0.0001 vs control group; ##P<0.01 vs model group.

图6 痛泻要方含药血清对5-HT刺激肝细胞SREBP-1/SCD1通路相关蛋白的影响

Fig.6 Effect of TXYF-medicated serum on expressions of the proteins in the SREBP-1/SCD1 pathway in 5-HT-stimulated cells （Mean±SD, n=3）. A: Expression level of SREBP-1 protein. B: Expression level of SCD1 protein. C： Expression level of 5-HT2AR protein. D： Protein bands of SREBP-1, SCD1 and 5-HT2AR. ***P<0.001 vs control group; #P<0.05, ##P<0.01 vs model group.

参考文献 40

[1]	Maiers JL, Chakraborty S. The cellular, molecular, and pathologic consequences of stress on the liver[J]. Am J Pathol, 2023, 193(10): 1353-4. doi：10.1016/j.ajpath.2023.07.003
[2]	Mooli RGR, Mukhi D, Ramakrishnan SK. Oxidative stress and redox signaling in the pathophysiology of liver diseases[J]. Compr Physiol, 2022, 12(2): 3167-92. doi：10.1002/j.2040-4603.2022.tb00210.x
[3]	Xu XY, Pang Y, Fan XQ. Mitochondria in oxidative stress, inflammation and aging: from mechanisms to therapeutic advances[J]. Signal Transduct Target Ther, 2025, 10(1): 190. doi：10.1038/s41392-025-02253-4
[4]	Liu SQ, Liu SS, He BY, et al. OXPHOS deficiency activates global adaptation pathways to maintain mitochondrial membrane potential[J]. EMBO Rep, 2021, 22(4): e51606. doi：10.15252/embr.202051606
[5]	周豪, 陈涛, 吴爱悯. 氧化应激对线粒体功能及椎间盘细胞的影响[J]. 四川大学学报: 医学版, 2024, 55(2): 249-55. doi：10.12182/20240360201
[6]	Wang SL, Long HJ, Hou LJ, et al. The mitophagy pathway and its implications in human diseases[J]. Signal Transduct Target Ther, 2023, 8(1): 304. doi：10.1038/s41392-023-01503-7
[7]	Lin JN, Chen XY, Du YY, et al. Mitophagy in cell death regulation: insights into mechanisms and disease implications[J]. Biomolecules, 2024, 14(10): 1270. doi：10.3390/biom14101270
[8]	Ho HJ, Shirakawa H. Oxidative stress and mitochondrial dysfunction in chronic kidney disease[J]. Cells, 2022, 12(1): 88. doi：10.3390/cells12010088
[9]	Zhao PZ, Shi WQ, Ye YF, et al. Atox1 protects hippocampal neurons after traumatic brain injury via DJ-1 mediated anti-oxidative stress and mitophagy[J]. Redox Biol, 2024, 72: 103156. doi：10.1016/j.redox.2024.103156
[10]	Yu HC, Jin L, Bai L, et al. C12ORF49 inhibits ferroptosis in hepatocellular carcinoma cells via reprogramming SREBP1/SCD1-mediated lipid metabolism[J]. Cell Death Discov, 2025, 11(1): 178. doi：10.1038/s41420-025-02480-2
[11]	Li N, Li XD, Ding YF, et al. SREBP regulation of lipid metabolism in liver disease, and therapeutic strategies[J]. Biomedicines, 2023, 11(12): 3280. doi：10.3390/biomedicines11123280
[12]	Martin AM, Young RL, Leong L, et al. The diverse metabolic roles of peripheral serotonin[J]. Endocrinology, 2017, 158(5): 1049-63. doi：10.1210/en.2016-1839
[13]	Erikci A, Ucar G, Yabanoglu-Ciftci S. Role of serotonin in the regulation of renal proximal tubular epithelial cells[J]. Ren Fail, 2016, 38(7): 1141-50. doi：10.1080/0886022x.2016.1194165
[14]	Ohta Y, Kosaka Y, Kishimoto N, et al. Convergence of the insulin and serotonin programs in the pancreatic β-cell[J]. Diabetes, 2011, 60(12): 3208-16. doi：10.2337/db10-1192
[15]	Nordlind K, Azmitia EC, Slominski A. The skin as a mirror of the soul: exploring the possible roles of serotonin[J]. Exp Dermatol, 2008, 17(4): 301-11. doi：10.1111/j.1600-0625.2007.00670.x
[16]	Ma YY, Liang XR, Li C, et al. 5-HT(2A) receptor and 5-HT degradation play a crucial role in atherosclerosis by modulating macrophage foam cell formation, vascular endothelial cell inflammation, and hepatic steatosis[J]. J Atheroscler Thromb, 2022, 29(3): 322-36. doi：10.5551/jat.58305
[17]	Ruddell RG, Oakley F, Hussain Z, et al. A role for serotonin (5-HT) in hepatic stellate cell function and liver fibrosis[J]. Am J Pathol, 2006, 169(3): 861-76. doi：10.2353/ajpath.2006.050767
[18]	Liu N, Sun SQ, Wang PJ, et al. The mechanism of secretion and metabolism of gut-derived 5-hydroxytryptamine[J]. Int J Mol Sci, 2021, 22(15): 7931. doi：10.3390/ijms22157931
[19]	Zhang YX, Li C, Liang XR, et al. Role of 5-HT degradation in acute liver injury induced by carbon tetrachloride[J]. Eur J Pharmacol, 2021, 908: 174355. doi：10.1016/j.ejphar.2021.174355
[20]	Sarkar S, Ghosh S, Biswas M. Naringin ameliorates high-fat diet-induced hepatotoxicity and dyslipidemia in experimental rat model via modulation of anti-oxidant enzymes, AMPK and SERBP-1c signaling pathways[J]. Toxicol Rep, 2025, 14: 102062. doi：10.1016/j.toxrep.2025.102062
[21]	李晶晶, 邓修洪, 杨霞, 等. 痛泻要方治疗大肠相关性疾病的研究进展[J]. 四川中医, 2025, 43(4): 214-21.
[22]	孙羽中, 陈富丽, 窦志芳. 痛泻要方对IBS肝郁脾虚证大鼠血清、结肠5-羟色胺的影响[J]. 山西中医学院学报, 2017(6): 20-3.
[23]	张丽敏, 赵清玉, 王一鸣, 等. 基于短链脂肪酸-5-羟色胺转化代谢机制研究痛泻要方对肠易激综合征模型肠道黏膜屏障的作用[J]. 中国药理学通报, 2025, 41(10): 1963-72.
[24]	罗运凤, 高洁, 柴艺汇, 等. 痛泻要方对肝郁脾虚型溃疡性结肠炎模型大鼠结肠SERT，肝脏5-HT2AR蛋白表达的影响[J]. 中国实验方剂学杂志, 2021, 27(2): 15-21. doi：10.13422/j.cnki.syfjx.20202103
[25]	Park J, Jeong W, Yun C, et al. Serotonergic regulation of hepatic energy metabolism[J]. Endocrinol Metab, 2021, 36(6): 1151-60. doi：10.3803/enm.2021.1331
[26]	Li X, Guo KK, Li T, et al. 5-HT 2 receptor mediates high-fat diet-induced hepatic steatosis and very low density lipoprotein overproduction in rats[J]. Obes Res Clin Pract, 2018, 12(): 16-28. doi：10.1016/j.orcp.2016.03.015
[27]	林陶秀, 张文娟, 张悦健, 等. 最佳中药含药血清浓度的筛选方法研究现状[J]. 中国实验方剂学杂志, 2023, 29(2): 195-202.
[28]	廖月, 何毅怀, 罗亚文. 氧化应激在急性肝损伤中的作用[J]. 临床肝胆病杂志, 2022, 38(10): 2402-7.
[29]	王颖, 王一方, 罗朝桂, 等. 脂肪肝中的线粒体氧化应激分子机制及营养素干预策略[J]. 中国科学: 生命科学, 2025, 55(1): 67-81.
[30]	Zhao SN, Guo Y, Yin XZ. Lipid peroxidation in ferroptosis and association with nonalcoholic fatty liver disease[J]. Front Biosci, 2023, 28(12): 332. doi：10.31083/j.fbl2812332
[31]	杨冰清, 尹静亚, 王琦. 肝线粒体功能障碍在非酒精性脂肪性肝病发病中的作用机制[J]. 临床肝胆病杂志, 2024, 40(1): 147-50. doi：10.12449/JCH240124
[32]	廖太阳, 李晓辰, 杨楠, 等. 白芍总苷经活性氧-线粒体途径改善骨关节炎软骨细胞自噬性死亡[J]. 医学研究与战创伤救治, 2023, 36(4): 337-42. doi：10.16571/j.cnki.2097-2768.2023.04.001
[33]	Liu J, Chen X, Yue CJ, et al. Effect of selenylation modification on immune-enhancing activity of Atractylodes macrocephala polysaccharide[J]. Int J Biol Macromol, 2015, 72: 1435-40. doi：10.1016/j.ijbiomac.2014.10.022
[34]	Liu HH, Xu Y, Li CJ, et al. An SCD1-dependent mechanoresponsive pathway promotes HCC invasion and metastasis through lipid metabolic reprogramming[J]. Mol Ther, 2022, 30(7): 2554-67. doi：10.1016/j.ymthe.2022.03.015
[35]	Lounis MA, Bergeron KF, Burhans MS, et al. Oleate activates SREBP-1 signaling activity in SCD1-deficient hepatocytes[J]. Am J Physiol Endocrinol Metab, 2017, 313(6): E710-20. doi：10.1152/ajpendo.00151.2017
[36]	Jin YY, Liu YC, Ge A, et al. Advances in fibroblast-based cardiac reprogramming in the treatment of heart disease[J]. Inflamm Res, 2025, 74(1): 114. doi：10.1007/s00011-025-02079-4
[37]	Eid W, Dauner K, Courtney KC, et al. mTORC1 activates SREBP-2 by suppressing cholesterol trafficking to lysosomes in mammalian cells[J]. Proc Natl Acad Sci USA, 2017, 114(30): 7999-8004. doi：10.1073/pnas.1705304114
[38]	毛国章, 卓立甬, 曹莞婷, 等. 基于 “肝-肠” 轴探讨痛泻要方治疗腹泻型肠易激综合征研究进展[J]. 中国中医药图书情报杂志, 2024, 48(5): 288-92.
[39]	张蜀, 吴至久, 范艺缤, 等. 痛泻要方颗粒对肝郁脾虚型腹泻型肠易激综合征患者的临床疗效[J]. 中成药, 2023, 45(10): 3509-12.
[40]	袁亚利, 李军祥, 毛堂友, 等. 胆汁酸代谢-TGR5轴调控腹泻型肠易激综合征的作用机制及中医药相关研究进展[J]. 世界科学技术-中医药现代化, 2022, 24(12): 4960-8.