社交应激性抑郁对小鼠肺癌的影响及淫羊藿苷干预的研究
摘要
目的以肿瘤生长转移为主要疗效指标,评价淫羊藿苷对肺癌伴抑郁的疗效。从直接抗抑郁、抗炎症、抗血管生成和抗肿瘤等环节,探讨淫羊藿苷的疗效机理。
方法1.建立肺癌伴抑郁模型,并采用淫羊藿苷干预。观察肿瘤重量及肺转移结节数目;免疫组化法测血管生成相关指标VEGF、CD31并行MVD计数,炎症相关指标CD11b;流式细胞术测免疫抑制相关指标MDSC;ELISA测VEGF和IL-6。2.建立社交应激性抑郁模型,并采用淫羊藿苷干预。MED动物行为分析仪记小鼠运动轨迹及社交区域停留时间;ELISA法测皮质酮、IL-6;放射性配基和western blot法测糖皮质激素受体(GR)。3.建立脂多糖炎症模型,采用淫羊藿苷及其代谢产物干预。ELISA法测炎症因子TNF-α、IL-6和PGE2;GriessReagent法测NO;western blot法测炎症相关酶iNOS和COX2;流式细胞术测CD11b。4.建立肺癌模型及血管新生模型,采用淫羊藿苷及其代谢产物干预。体外部分:CCK-8试剂盒测其对小鼠肺癌细胞株(LLC)细胞毒作用;流式细胞术测LLC细胞周期;流式细胞术和Tunel法测LLC调亡;HUVEC划痕闭合实验和鸡胚绒毛尿囊膜实验(CAM)评价抗血管生成疗效。体内部分:观察肿瘤重量及肺转移结节数目;免疫组化法测VEGF、CD31并行MVD计数;流式细胞术测MDSC;ELISA法测VEGF和IL-6。结果:1.肺癌伴抑郁模型的肿瘤生长转移明显增加,淫羊藿苷则显示了干预作用,同时可降低MDSC、CD11b、IL-6、VEGF及MVD水平。2.淫羊藿苷可增加社交应激性抑郁模型社交区域活动时间,同时降低皮质酮及IL-6水平,部分恢复GR蛋白表达和结合活性。3.淫羊藿苷及其代谢产物可抑制脂多糖炎症模型TNF-α、IL-6、PGE2、NO、CD11b水平,同时降低iNOS和COX2表达。4.淫羊藿苷代谢产物(淫羊藿次苷Ⅱ)对LLC细胞株显示细胞毒作用、周期阻滞和促凋亡作用;淫羊藿苷代谢产物(淫羊藿素)显示了抗血管生成作用;淫羊藿苷体内可抑制肿瘤生长转移。结论:1.社交应激性抑郁可促进小鼠肿瘤生长转移,同时伴免疫抑制、炎症加重和肿瘤血管新生增强。淫羊藿苷能部分逆转上述进程。2.淫羊藿苷可通过直接抗抑郁、抗炎症、抗肿瘤、抗血管生成和提高免疫等多环节对肿瘤伴抑郁干预。3.淫羊藿素和淫羊藿次苷Ⅱ可能是淫羊藿苷干预肺癌伴抑郁的体内主要效应物质。
Objective: Evaluate icariin's effect on tumor growth and metastasis of tumor and depression coexistence model. Explore icariin's intervention targets on tumor and depression coexistence model. Method: 1. Tumor and depression coexistence model were treated with icariin. Tumor weight and lung metastatic nodules were calculated; Tumor tissue's VEGF, CD11b and MVD were tested by immunohistochemistry; MDSC were measured by flow cytometry; Serum VEGF and IL-6 were detceted by ELISA. 2. Social stress-induced depression model were treated with icariin .The residence time at interation zone was recorded by MED behavior analysis system; Serum corticosterone and IL-6 were tested by ELISA; GR was detected by radioative ligand method and western blot. 3. Inflammation model stimulated by lipopolysaccharide were treated with icariin and its metabolites (icaritin and icariside) . TNF-α, IL-6 and PGE2 were measured by ELISA; NO was detected by Griess Reagent method. iNOS and COX2 were tested by western blot; CD11b were measured by flow cytometry. 4. LLC tumor model and angiogenesis model were treated with icariin and its metabolites . In vitro experiment: LLC cell proliferation was tested by cell counting kit-8; LLC cell cycle was measured by flow cytometry; Apoptosis was observed by Tunel method; HUVEC and CAM were used as angiogenesis models to evaluate anti-angiogenesis effect .In vivo experiment: Tumor weight and lung metastasis nodules were calculated; Tumor tissue's VEGF and MVD were tested by immunohistochemistry; MDSC was measured by flow cytometry ; Serum VEGF and IL-6 were detected by ELISA. Results: 1. Social stress induced depression promoted tumor growth and metastasis. Icariin treated this tumor and depression coexistence model effectively. It reduced the MDSC, CD11b, IL-6, VEGF and MVD levels. 2. Icariin treated social stress model effectively .It increased depression mice residence time at interation zone, reduced the level of corticosterone and IL-6, and restored GR protein expression and binding activity. 3. Icariin and its metabolity ( icaritin) inhibited the inflammatory model stimulated by LPS effectively. It reduced TNF-α, IL-6, PGE2, NO, CD11b levels and expression of iNOS and C0X2. 4. Icarisde displayed cytotoxicity, cycle arrest and apoptosis effect on LLC, while icaritin showed anti-angiogenesis effect in vitro. Icariin can inhibit tumor growth and metastasis in vivo effectively. Conclusion: 1. Social stress-induced depression can promote tumor growth and metastasis dramatically, and exacerbate the immune suppression,inflammation and angiogenesis. Icariin can intervene the above process .2. Icariin displayed anti-depression ,anti- inflammatory, anti-tumor effects, respectively. These effect can benefit cancer patients with depression. 3. Icarisde and icaritin may be the major in vivo active ingredients of icariin.
方法1.建立肺癌伴抑郁模型,并采用淫羊藿苷干预。观察肿瘤重量及肺转移结节数目;免疫组化法测血管生成相关指标VEGF、CD31并行MVD计数,炎症相关指标CD11b;流式细胞术测免疫抑制相关指标MDSC;ELISA测VEGF和IL-6。2.建立社交应激性抑郁模型,并采用淫羊藿苷干预。MED动物行为分析仪记小鼠运动轨迹及社交区域停留时间;ELISA法测皮质酮、IL-6;放射性配基和western blot法测糖皮质激素受体(GR)。3.建立脂多糖炎症模型,采用淫羊藿苷及其代谢产物干预。ELISA法测炎症因子TNF-α、IL-6和PGE2;GriessReagent法测NO;western blot法测炎症相关酶iNOS和COX2;流式细胞术测CD11b。4.建立肺癌模型及血管新生模型,采用淫羊藿苷及其代谢产物干预。体外部分:CCK-8试剂盒测其对小鼠肺癌细胞株(LLC)细胞毒作用;流式细胞术测LLC细胞周期;流式细胞术和Tunel法测LLC调亡;HUVEC划痕闭合实验和鸡胚绒毛尿囊膜实验(CAM)评价抗血管生成疗效。体内部分:观察肿瘤重量及肺转移结节数目;免疫组化法测VEGF、CD31并行MVD计数;流式细胞术测MDSC;ELISA法测VEGF和IL-6。结果:1.肺癌伴抑郁模型的肿瘤生长转移明显增加,淫羊藿苷则显示了干预作用,同时可降低MDSC、CD11b、IL-6、VEGF及MVD水平。2.淫羊藿苷可增加社交应激性抑郁模型社交区域活动时间,同时降低皮质酮及IL-6水平,部分恢复GR蛋白表达和结合活性。3.淫羊藿苷及其代谢产物可抑制脂多糖炎症模型TNF-α、IL-6、PGE2、NO、CD11b水平,同时降低iNOS和COX2表达。4.淫羊藿苷代谢产物(淫羊藿次苷Ⅱ)对LLC细胞株显示细胞毒作用、周期阻滞和促凋亡作用;淫羊藿苷代谢产物(淫羊藿素)显示了抗血管生成作用;淫羊藿苷体内可抑制肿瘤生长转移。结论:1.社交应激性抑郁可促进小鼠肿瘤生长转移,同时伴免疫抑制、炎症加重和肿瘤血管新生增强。淫羊藿苷能部分逆转上述进程。2.淫羊藿苷可通过直接抗抑郁、抗炎症、抗肿瘤、抗血管生成和提高免疫等多环节对肿瘤伴抑郁干预。3.淫羊藿素和淫羊藿次苷Ⅱ可能是淫羊藿苷干预肺癌伴抑郁的体内主要效应物质。
Objective: Evaluate icariin's effect on tumor growth and metastasis of tumor and depression coexistence model. Explore icariin's intervention targets on tumor and depression coexistence model. Method: 1. Tumor and depression coexistence model were treated with icariin. Tumor weight and lung metastatic nodules were calculated; Tumor tissue's VEGF, CD11b and MVD were tested by immunohistochemistry; MDSC were measured by flow cytometry; Serum VEGF and IL-6 were detceted by ELISA. 2. Social stress-induced depression model were treated with icariin .The residence time at interation zone was recorded by MED behavior analysis system; Serum corticosterone and IL-6 were tested by ELISA; GR was detected by radioative ligand method and western blot. 3. Inflammation model stimulated by lipopolysaccharide were treated with icariin and its metabolites (icaritin and icariside) . TNF-α, IL-6 and PGE2 were measured by ELISA; NO was detected by Griess Reagent method. iNOS and COX2 were tested by western blot; CD11b were measured by flow cytometry. 4. LLC tumor model and angiogenesis model were treated with icariin and its metabolites . In vitro experiment: LLC cell proliferation was tested by cell counting kit-8; LLC cell cycle was measured by flow cytometry; Apoptosis was observed by Tunel method; HUVEC and CAM were used as angiogenesis models to evaluate anti-angiogenesis effect .In vivo experiment: Tumor weight and lung metastasis nodules were calculated; Tumor tissue's VEGF and MVD were tested by immunohistochemistry; MDSC was measured by flow cytometry ; Serum VEGF and IL-6 were detected by ELISA. Results: 1. Social stress induced depression promoted tumor growth and metastasis. Icariin treated this tumor and depression coexistence model effectively. It reduced the MDSC, CD11b, IL-6, VEGF and MVD levels. 2. Icariin treated social stress model effectively .It increased depression mice residence time at interation zone, reduced the level of corticosterone and IL-6, and restored GR protein expression and binding activity. 3. Icariin and its metabolity ( icaritin) inhibited the inflammatory model stimulated by LPS effectively. It reduced TNF-α, IL-6, PGE2, NO, CD11b levels and expression of iNOS and C0X2. 4. Icarisde displayed cytotoxicity, cycle arrest and apoptosis effect on LLC, while icaritin showed anti-angiogenesis effect in vitro. Icariin can inhibit tumor growth and metastasis in vivo effectively. Conclusion: 1. Social stress-induced depression can promote tumor growth and metastasis dramatically, and exacerbate the immune suppression,inflammation and angiogenesis. Icariin can intervene the above process .2. Icariin displayed anti-depression ,anti- inflammatory, anti-tumor effects, respectively. These effect can benefit cancer patients with depression. 3. Icarisde and icaritin may be the major in vivo active ingredients of icariin.
引文
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2. Thompson DS, Shear MK. Psychiatric disorders and gynecological oncology: a review of the literature. Gen Hosp Psychiatry, 1998, 20(4): 241-7.
3. V Strong, R Waters.Emotional distress in cancer patients: the Edinburgh Cancer Centre symptom study[J].British Journal of Cancer.2007,96, 868-874.
4. Stefanek, M. & McDonald. in Handbook of Behavioral Science and Cancer.American Psychological Association, Washington DC)
5. Reiche, E. M., Nunes, S. O. Stress, depression, the immune system, and cancer. Lancet Oncol. 2004,5,617-625.
6. Spiegel, D. & Giese-Davis. Depression and cancer: mechanisms and disease progression. Biol. Psychiatry ,2003.54,269-282.
7. Price, M. A. et al. The role of psychosocial factors in the development of breast carcinoma: part I. The cancer prone personality. Cancer 2001,91, 679-685.
8. Lillberg, K. et al. Stressful life events and risk of breast cancer in 10808 women: a cohort study. Am. J.Epidemiol.2003,157,415-423.
9. Newport DJ, Nemeroff CB. Assessment and treatment of depression in the cancer patient. J Psychosom Res, 1998,45(3): 215-37.
10. Watson M , H aviland JS.Influence ofpsychologicalresponse on survivalin breast cancer:a population based cohort study. Lancet,1999,354(9187):1331-1336.
11. Chubak J, Buist DS. Breast cancer recurrence risk in relation to antidepressant use after diagnosis.Breast Cancer Res Treat. 2007
12. Tamim HM, Mahmud S. Antidepressants and risk of prostate cancer: a nested case - control study. Prostate Cancer Prostatic Dis. 2008 ,11(1):53-60.
13. Toh S, Rodriguez LA. Use of antidepressants and risk of lung cancer. Cancer Causes Control. 2007,18(10):1055-64.
14. Brandes LJ, Arron RJ, Stimulation of malignant growth in rodents by antidepressant drugs at clinically relevant doses. Cancer Res. 1992,52(13):3796-800.
15. Herbert TB, Cohen S.Stress and immunity in humans: A meta-analytic review. Psychosomatic Med,1993,55:364-379.
16. Gruber BL, Hersh SP. Immunological responses of breast cancer patients to behavioral interventions.Biofeedback Self Regul, 1993,18 (1):1-22
17. Yoko Ishihara, Kenichi Matsunaga.Time -dependent effects of stressor application on metastasis of tumor cells in the lung and its regulation by an immunomodulator in mice Psychoneuroendocrinology .1999,24:713-726
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