Research on Effective Fraction and Mechanism of Elaphurus Davidiauus Cornu Improving Depression-Like Behaviors on CUMS Mice via Regulation of Neuronal Apoptosis in Hippocampus
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摘要:
目的 基于慢性不可预见性动物应激(CUMS)模型考察麋鹿角各提取物的抗抑郁效用, 以及其对氧化损伤诱导神经元凋亡的抑制效应, 并对产生该效应的功效物质进行筛选研究。 方法 制备麋鹿角各提取物, 采用小鼠CUMS模型, 通过糖水偏嗜实验、悬尾实验和强迫游泳实验评价麋鹿角提取物的抗抑郁功效。采用TUNEL法检测小鼠海马区细胞凋亡情况, 采用相应试剂盒测定小鼠血清中丙二醛(MDA)的含量，过氧化氢酶(CAT)、超氧化物歧化酶(SOD)及谷胱甘肽还原酶(GSH)的活性。采用小鼠海马神经元细胞系HT22和大鼠肾上腺嗜铬瘤细胞系PC12, 以过氧化氢(H2O2)作为造模剂构建氧化损伤模型, 通过MTT法评价麋鹿角提取物对细胞氧化损伤的保护作用。采用TUNEL染色法考察麋鹿角水提取物(EDC-WE)对H2O2诱导HT22细胞凋亡的抑制作用, 进一步采用Western blot法考察神经元细胞凋亡通路相关蛋白Caspase3、Cleaved-Caspase3、Caspase9、Cleaved-Caspase9的表达水平。将EDC-WE采用超滤法分离为分子量>100 kD、分子量30~100 kD、分子量10~30 kD、分子量 < 10 kD的4个组分, 通过MTT法评价EDC-WE不同分子量段对HT22细胞氧化损伤的保护作用, 并对分子量 < 10 kD的组分进行TUNEL染色分析和Caspase信号通路的考察验证。 结果 麋鹿角各提取物均能显著上调模型小鼠糖水偏嗜率, 缩短小鼠悬尾和强迫游泳不动时间, 在各提取物中以EDC-WE的效用最强。EDC-WE能够对抗HT22细胞的氧化损伤, 并调控Caspase信号通路对抗H2O2诱导的HT22细胞凋亡, 其中分子量 < 10 kD的麋鹿角水溶性物质抗细胞氧化损伤效用较强。 结论 EDC-WE可以抑制氧化应激诱导的神经元细胞凋亡, 从而实现抗抑郁作用, 其中分子量 < 10 kD的麋鹿角水溶性物质可能是关键的功效成分。 Abstract:OBJECTIVE To investigate the effect of Elaphurus davidiauus Cornu (EDC) on anti-depressive effect via chronic unexpected mild stress (CUMS) model and to locate EDC efficient substances with the effect of inhibiting the oxidative damage induced neuronal apoptosis. METHODS EDC extracts were prepared, and the anti-depression effect of EDC extracts was evaluated by sucrose preference test, tail suspension test and forced swimming test in CUMS mice model. The expression of malondialdehyde (MDA) and the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GSH) in the serum of mice were determined by related detection kits. The mouse hippocampal neuronal cell line (HT22) and rat adrenal pheochromocytoma cells (PC12) were used to construct an oxidative injury model with hydrogen peroxide. The protective effect of EDC on the oxidative injury was evaluated by MTT assay. TUNEL staining was used to investigate the anti-apoptosis effect of a water extract from EDC (EDC-WE) on HT22 cells after oxidative damage. The expression of neuronal apoptosis pathway-related proteins, such as Caspase3, Cleaved-caspase3, Caspase9 and Cleaved-caspase9 were detected by Western blotting. The EDC-WE was further separated into four fractions with molecular weight more than 100 kD, molecular weight 30~100 kD, molecular weight 10~30 kD and molecular weight less than 10 kD by ultrafiltration. The protective effect of different molecular weight segments of EDC-WE on oxidative damage in HT22 cells was evaluated by the MTT method. The effect of the anti-apoptosis effect on components with molecular weight less than 10 kD was analyzed by TUNEL staining and its mechanism of anti-apoptosis via detecting the Caspase signaling pathway by Western blot. RESULTS EDC extracts could significantly up-regulate the sucrose preference rate of model mice, shorten the immobile time of tail suspension and forced swimming test, and EDC-WE showed the strongest effect among the three extracts. EDC-WE could prevent the hydrogen peroxide oxidative damage induced HT22 cell apoptosis via regulating the Caspase signaling pathway. The water-soluble substance of EDC with molecular weight less than 10 kD had the strongest effect on protecting against cell oxidative damage among the four components. CONCLUSION The anti-depressive effect of EDC-WE is related to its effect on inhibiting the oxidation induced apoptosis of neuronal cells via regulating the Caspase signaling pathway, and the water-soluble substance of EDC with molecular weight less than 10 kD may be the key effective component responsible for the anti-depressive effect. -
Key words:
- Elaphurus davidiauus Cornu /
- oxidative stress /
- depression /
- apoptosis /
- functional substance
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图 1 麋鹿角各提取物对CUMS模型小鼠抗抑郁样行为学评价
注: 与空白组比较, ##P < 0.01;与CUMS组比较, *P < 0.05, * *P < 0.01。
Figure 1. The anti-depression effect of EDC extract via behavior test in CUMS mice model
图 2 麋鹿角提取物对CUMS模型小鼠血清中过氧化物及抗氧化酶表达的影响
注: 与空白组比较, ##P < 0.01;与CUMS组比较, *P < 0.05, * *P < 0.01。
Figure 2. The effect of EDC extract on regulating the expression of peroxides and antioxidant enzymes in CUMS mice model
图 3 麋鹿角各提取物对CUMS模型小鼠海马神经元细胞凋亡的影响(×200)
注: 与空白组比较, ##P < 0.01;与模型组比较, * *P < 0.01。
Figure 3. The effect of EDC extract on preventing hippocampus neuron apoptosis in CUMS mice model (×200)
图 4 麋鹿角各提取物对HT22和PC12细胞氧化损伤的保护作用
注: 与空白组比较, #P < 0.05, ##P < 0.01;与模型组比较, *P < 0.05, * *P < 0.01。
Figure 4. The effect of EDC extract on preventing HT22 and PC12 cells from oxidative damage
图 5 麋鹿角各提取物对氧化损伤诱导HT22细胞凋亡的影响
注: 与空白组比较, ##P < 0.01;与模型组比较, *P < 0.05, * *P < 0.01。
Figure 5. The effect of EDC extract on preventing HT22 from apoptosis induced by oxidative damage
图 6 EDC-WE不同分子量部位对HT22细胞氧化损伤的保护作用
注: 与空白组比较, ##P < 0.01;与模型组比较, *P < 0.05, * *P < 0.01。
Figure 6. The anti-oxidative effect of HT22 cells by different molecular weight segments of EDC-WE
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[1] 陶弘景. 名医别录辑校本[M]. 尚志钧, 辑校. 北京: 中国中医药出版社, 2013: 142-143.TAO HJ. Miscellaneous Records of Famous Physicians[M]. SHANG ZJ, annotated. Beijing: China press of traditional Chinese medicine Co., Ltd., 2013: 142-143. [2] 国家中医药管理局《中华本草》编委会. 中华本草[M]. 上海: 上海科学技术出版社, 2017: 665-667.Editorial Board of Chinese Materia Medica, State Administration of Traditional Chinese Medicine. Chinese Materia Medica[M]. Shanghai: Shanghai scientific & techical publishers, 2017: 665-667. [3] 孙思邈. 备急千金要方校释[M]. 李景荣, 等校释. 北京: 人民卫生出版社, 1998: 432-433.SUN SM, The annotation of Important Formulas Worth a Thousand Gold Pieces for Emergency [M]. LI JQ, et. al annotated. Beijing: People's medical publishing house, 1998: 432-433. [4] 张璐. 本经逢源[M]. 太原: 山西科学技术出版社, 2015: 305-306.ZHANG L. Encounting the Sources of the Classic of Materia Medica [M]. Taiyuan: Shanxi scientific & technical publishers, 2015: 305-306. [5] 刘梦秋, 曹程, 曲苏晨, 等. 麋鹿角调控慢性压力应激小鼠神经营养因子和压力应激因子抗抑郁作用研究[J]. 世界科学技术-中医药现代化, 2019, 21(9): 1805-1812. https://www.cnki.com.cn/Article/CJFDTOTAL-SJKX201909007.htmLIU MQ, CAO C, QU SC, et al. Researches on anti-depression mechanism of elk antler via regulation of neurotrophic factors and stress factors on chronic unpredictable mild stress induced mice[J]. Mod Tradit Chin Med Mater Med World Sci Technol, 2019, 21(9): 1805-1812. https://www.cnki.com.cn/Article/CJFDTOTAL-SJKX201909007.htm [6] SLAVICH GM, IRWIN MR. From stress to inflammation and major depressive disorder: A social signal transduction theory of depression[J]. Psychol Bull, 2014, 140(3): 774-815. doi: 10.1037/a0035302 [7] CIPRIANI A, FURUKAWA TA, SALANTI G, et al. Comparative efficacy and acceptability of 21 antidepressant drugs for the acute treatment of adults with major depressive disorder: A systematic review and network meta-analysis[J]. Lancet, 2018, 391(10128): 1357-1366. doi: 10.1016/S0140-6736(17)32802-7 [8] ROTENSTEIN LS, RAMOS MA, TORRE M, et al. Prevalence of depression, depressive symptoms, and suicidal ideation among medical students[J]. JAMA, 2016, 316(21): 2214. doi: 10.1001/jama.2016.17324 [9] GOLDSTEIN BI, CARNETHON MR, MATTHEWS KA, et al. Major depressive disorder and bipolar disorder predispose youth to accelerated atherosclerosis and early cardiovascular disease: A scientific statement from the American heart association[J]. Circulation, 2015, 132(10): 965-986. doi: 10.1161/CIR.0000000000000229 [10] GRAHEK I, SHENHAV A, MUSSLICK S, et al. Motivation and cognitive control in depression[J]. Neurosci Biobehav Rev, 2019, 102: 371-381. doi: 10.1016/j.neubiorev.2019.04.011 [11] JIN Y, SUN LH, YANG W, et al. The role of BDNF in the neuroimmune axis regulation of mood disorders[J]. Front Neurol, 2019, 10: 515. doi: 10.3389/fneur.2019.00515 [12] KOJIMA M, MATSUI K, MIZUI T. BDNF pro-peptide: Physiological mechanisms and implications for depression[J]. Cell Tissue Res, 2019, 377(1): 73-79. doi: 10.1007/s00441-019-03034-6 [13] BAO AM, SWAAB DF. The human hypothalamus in mood disorders: The HPA axis in the center[J]. IBRO Rep, 2018, 6: 45-53. [14] PARK C, ROSENBLAT JD, BRIETZKE E, et al. Stress, epigenetics and depression: A systematic review[J]. Neurosci Biobehav Rev, 2019, 102: 139-152. doi: 10.1016/j.neubiorev.2019.04.010 [15] DREVETS WC, WITTENBERG GM, BULLMORE ET, et al. Immune targets for therapeutic development in depression: Towards precision medicine[J]. Nat Rev Drug Discov, 2022, 21(3): 224-244. doi: 10.1038/s41573-021-00368-1 [16] OTTE C, GOLD SM, PENNINX BW, et al. Major depressive disorder[J]. Nat Rev Dis Primers, 2016, 2: 16065. doi: 10.1038/nrdp.2016.65 [17] ZHU KY, XU SL, CHOI RCY, et al. Kai-Xin-San, a Chinese herbal decoction containing ginseng radix et rhizoma, polygalae radix, acori tatarinowii rhizoma, and poria, stimulates the expression and secretion of neurotrophic factors in cultured astrocytes[J]. Evid Based Complement Alternat Med, 2013, 2013: 731385. [18] 张月月, 武占娟, 王君明, 等. 地黄及其传统复方防治抑郁症研究[J]. 中华中医药学刊, 2021, 39(12): 137-140. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYHS202112032.htmZHANG YY, WU ZJ, WANG JM, et al. Research on Dihuang(rehmanniae Radix) and its traditional prescriptions in prevention and treatment of depression[J]. Chin Arch Tradit Chin Med, 2021, 39(12): 137-140. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYHS202112032.htm [19] 畅洪昇, 鲁艺, 王伟明, 等. 何首乌的抗抑郁作用及其对海马5HT1A受体表达和神经细胞发生的影响[J]. 北京中医药大学学报, 2012, 35(12): 822-825. https://www.cnki.com.cn/Article/CJFDTOTAL-JZYB201212009.htmCHANG HS, LU Y, WANG WM, et al. Anti-depression effect of Heshouwu (Radix Polygoni Muotiflori) and its influences on expression of hippocampus 5HT1A receptor and neurogenesis[J]. J Beijing Univ Tradit Chin Med, 2012, 35(12): 822-825. https://www.cnki.com.cn/Article/CJFDTOTAL-JZYB201212009.htm [20] 魏婷, 何先元, 朱潇宏, 等. 九制黄精及其配伍对CUMS抑郁模型小鼠物质能量代谢的影响[J]. 食品与机械, 2022, 38(7): 180-185, 207. https://www.cnki.com.cn/Article/CJFDTOTAL-SPJX202207029.htmWEI T, HE XY, ZHU XH, et al. Comparative study of processed Polygonatum cyrtonema and its compatibility affecting on material and energy metabolism in CUMS depressed model mice[J]. Food & Mach, 2022, 38(7): 180-185, 207. https://www.cnki.com.cn/Article/CJFDTOTAL-SPJX202207029.htm [21] 李锋涛, 段金廒, 钱大玮, 等. 麋鹿角对D-半乳糖诱导小鼠衰老模型抗衰老作用[J]. 南京中医药大学学报, 2014, 30(3): 235-238. http://xb.njucm.edu.cn/article/id/zr201403011LI FT, DUAN JA, QIAN DW, et al. Effect of elaphuri davidiani Cornu on the aging model induced by D-galactose in mice[J]. J Nanjing Univ Tradit Chin Med, 2014, 30(3): 235-238. http://xb.njucm.edu.cn/article/id/zr201403011 [22] 李锋涛. 麋鹿角功效物质基础研究[D]. 南京: 南京中医药大学, 2014.LI FT. Study on material foundation of traditional efficacies of elaphuri davidiani Cornu[D]. Nanjing: Nanjing University of Chinese Medicine, 2014. [23] 杨晓男, 阮丽君, 江兴, 等. 多肽类药物与中药肽研究展望[J]. 中国中药杂志, 2022, 47(22): 5978-5990. https://www.cnki.com.cn/Article/CJFDTOTAL-ZGZY202222002.htmYANG XN, RUAN LJ, JIANG X, et al. Overview of research and development of polypeptide drugs and traditional Chinese medicine-peptides[J]. China J Chin Mater Med, 2022, 47(22): 5978-5990. https://www.cnki.com.cn/Article/CJFDTOTAL-ZGZY202222002.htm -
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