前列通瘀汤对实验性慢性非细菌性前列腺大鼠的治疗作用和机制研究
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摘要
慢性非细菌性前列腺炎/慢性盆腔疼痛综合征(Chronic pelvic pain syndrome, CPPS),是男科常见疾病之一,主要表现为骨盆生殖区的疼痛和排尿异常,其病因及㎜研究显示,CPPS是多种因素共同作用的结果,其中自身免疫反应起着重要的作用,而这种作用是通过炎性细胞因子介导的。中医辨证论治,有一定的优势,已成为临床治疗本病的重要方法之一。前列通瘀汤具有活血化瘀、清热利湿的功效,治疗CPPS能明显缓解疼痛症状,取得了较好的疗效。
研究显示,前列腺素E2(Prostaglandin-E2,PGE2)在CPPS患者的前列腺液(expressed prostatic secretions, EPS)中明显升高,是引起疼痛的主要原因。组织损伤、炎症和局部感染等使白细胞介素-1β(interleukin-1β,IL-1β)产生增多,继而诱导环氧化酶-2(Cyclooxygenase-2,COX-2)的基因表达,COX-2的产生迅速增多,促使细胞产生大量的PGE2,高浓度的PGE2又能抑制β-內啡肽(β-endorphin,β-EP)的分泌和调节疼痛的功能。应用特异性COX-2抑制剂治疗CPPS可明显减轻疼痛。我们推测前列通瘀汤可能是通过降低PGE2而对CPPS产生治疗作用。本课题旨在通过动物实验研究,探讨前列通瘀汤对PGE2等细胞因子的干预作用,揭示其治疗CPPS的可能机制。
目的本课题采用免疫佐剂法制作实验性CPPS大鼠模型,观察前列通瘀汤对实验大鼠前列腺组织形态学的影响,评价其疗效;以COX-2抑制剂塞来昔布为阳性对照药,观察前列通瘀汤对致痛因子PGE2,及与之密切相关的IL-1β、COX-2和β-EP的影响,探讨前列通瘀汤治疗CPPS的可能机理,并总结其组方规律和用药特点,为指导临床合理选择有效的中药及方剂提供思路。
方法取3月龄180-220g雄性Wistar种大鼠62只,随机分为正常组10只、实验组52只。实验组大鼠采用同种系大鼠前列腺蛋白提纯液,辅以双重免疫佐剂方法,复制实验性CPPS大鼠模型。模型成功后,再随机将实验组大鼠分为模型组、塞来昔布组、前列通瘀汤大、中、小剂量组五组。前列通瘀汤大、中、小剂量组给药剂量分别为32、16、8g生药/kg。塞来昔布组给药剂量为0.04 g/kg。正常组和模型组给于相同剂量的蒸馏水。以上各组均灌胃给药,每日一次,连续给药30d。第30天将各组大鼠称重后脱颈处死。取前列腺组织,观察其病理改变用ELISA法测定前列腺组织匀浆IL-1β、COX-2、PGE2、β-EP的含量。
结果①病理观察显示,模型组大鼠前列腺组织表现为慢性炎症的病理变化。其前列腺腺体普遍与周围组织粘连,腺体呈暗红色或棕黄色,质稍硬,或有明显的硬结。显微镜下主要表现为淋巴细胞和单核细胞的浸润,间质水肿及纤维组织增生,前列腺上皮破坏、萎缩及脱落,前列腺管壁的增生或破坏。对前列腺病理切片的分级进行统计分析,显示模型组大鼠与正常大鼠前列腺组织病理表现差异有统计学意义。表明造模成功。②前列通瘀汤大剂量组和塞来昔布组大鼠前列腺上皮组织恢复正常,管壁结构完整,间质水肿消失。前列通瘀汤中剂量组和小剂量组对实验大鼠前列腺组织结构的破坏虽然也有一定程度的改善,但作用较轻。病理分级统计结果显示:前列通瘀汤大、中、小剂量组、塞来昔布组,与模型组比较差异均有统计学意义;前列通瘀汤小剂量组与正常组比较差异有统计学意义,其余各组与正常组比较,差异无统计学意义。③前列通瘀汤大、中、小剂量组,及塞来昔布组大鼠前列腺组织内PGE2及IL-1β、COX-2的水平较模型组明显降低;β-EP的水平较模型组明显升高。其中前列通瘀汤大剂量组与塞来昔布对照组比较,差异无统计学意义。
结论①采用自身免疫反应所造成的CPPS动物模型,其病理改变与临床的病理改变最为接近,发病机理也与人的发病较为符合,是一种较为理想的的造模方式。②前列通瘀汤能够减轻或消除模型大鼠前列腺组织的炎症反应,改善其组织结构的破坏,抗纤维化,对前列腺组织起到明显保护作用。③前列通瘀汤能够降低模型大鼠前列腺组织内PGE2及IL-1β、COX-2的水平,减轻前列腺组织炎症反应的程度,减轻盆腔生殖区域的疼痛;并能升高β-EP的水平,使局部抑制疼痛的作用加强。
研究表明,前列通瘀汤对CPPS的治疗作用可能与前列通瘀汤能调节前列腺组织的细胞因子含量,减轻前列腺组织炎症反应,对前列腺组织起到保护作用密切相关。
Chronic non-bacterial prostatitis/chronic pelvic pain syndrome (CPPS) is a common disease of male reproductive system, mainly to the reproductive pelvic pain and abnormal urination.The etiology and the pathogenesis of CPPS are not yet clear by now. Because of the high incidence of the disease, lack of typical symptoms, and difficult to treat, CPPS seriously impacts on the quality of life in patients. Recently studies have shown that CPPS is the result of a variety of factors working together, and autoimmune reactions play an important role in the pathogenesis, which is mediated by inflammatory cytokines. Traditional Chinese Medicine has some advantages in the treatment of chronic prostatitis by treatment based on syndrome differentiation, and already became one of the important ways of the clinical treatment. Qianlietongyu-tang is a Chinese medicine that can promote blood flow, relieve fever and promote urination. It can achieve a significant effect in treating CPPS, especially to relieve pain.
Research shows that prostaglandin E2 (PGE2) in CPPS prostatic fluid was significantly increased, and is the main cause of pain. Tissue injury, inflammation, and local infection, etc can cause increased production of interleukin-1 (IL-1). Increased IL-1 can induce the expression of cyclooxygenase-2 (COX-2) gene, resulting in a rapidly increase of COX-2, which induces a high production of PGE2 in the cells。High concentrations of PGE2 can inhibit the secretion and the analgesic effect ofβ-endorphin (β-EP). Specific COX-2 inhibitors can significantly reduce the pain of CPPS. We have conjectured that the possible treatment mechanism of Qianlietongyu-tang may be achieved by the way of decreasing the concentration of PGE2 of the prostate tissue. The topic made use of animal experiments to observe the intervention of Qianlietongyu-tang on cytokine such as PGE2, etc, and revealed its possible mechanism of treatment of CPPS.
Object The project made the experimental CPPS rat model by using SC purified prostate protein twice with immune adjuvant, observed the influence of Qianlietongyu-tang on morphological peculiarities of experimental rats prostate tissue, and evaluated its efficacy. The project observed the change of pain factor—PGE2, and the other cytokines as IL-1, COX-2 andβ2-EP that were closely related with PGE2, to explore the possible mechanism of treatment by Qianlietongyu-tang. The project also summarized the patterns of prescription and the characteristics of the drug to provide ideas for guiding reasonable choice of effective medicines and prescriptions in clinic.
Methods Selection of 62 male, 3-month-old, weight 180-220g Wistar rats, randomly divided into two groups: a control group, 10 rats and a model group, 52 rats. The model group was made by DPT vaccine in peritoneal injection, and rat prostatein purification of FCA emulsion multiple intra-dermal injections. The injected rats were again randomly divided into a control group, a model group, a celecoxib group and a group of low, middle and high dose of Qianlietongyu-tang. The low, middle and high dose group was given dose according to standards of 32,16,8g crude drug/kg respectively. The Celecoxib group administrates the dose of 0.04/kg. The control group and the model group were given the same dose of distilled water. The above groups were successively administrated oral for 30days, qd. All animals were killed 30d after treatment, and prostate glands were obtained to observe the histomorphological changes and detect the contents of IL-1β, COX-2, PGE2 andβ-endorphin in prostate-homogenate by enzyme linked immunosorbent assay.
Results①pathological findings: the model group shows chronic inflammation of prostate glandular tissue. The glandular organ of prostate gland adheres to the surrounding tissue, kermesinus or yellow, slightly hard in quality, or obviously indurate. Under microscope, infiltration of lymph and mononuclear cells, interstitial edema and fibroplasias, damage, atrophia, or ablate of prostate gland endepidermis, accrementition or damage of prostatid ducts can be seen. According to statistical analysis on classified pathological sections of prostate gland, the difference between model group and normal group has statistical significance. The above results indicate that the model building is successful.②In the high dose group of Qianlietongyu-tang and Celecoxib group, the epithelial tissues recover, the architecture of epithelial tissues are integrated, and the interstitial edemas disappear. In the mid and low dose group, the damage of prostate gland tissues recover slightly. According to classified statistical analysis, the differences among each group of the Qianlietongyu-tang and the Celecoxib group had statistical significance. The difference between the low dose group of Qianlietongyu-tang and normal group had statistical significance, whereas the differences among the other groups had not.③Compared to the model group, the level of PGE2, IL-1βand COX-2 of prostate glandular tissues reduced in each group of Qianlietongyu-tang and Celecoxib, whereasβ-EP increased obviously. The difference between high dose group of Qianlietongyu-tang and Celecoxib group had no statistical significance.
Conclusion①Using the way of autoimmune reaction to induce the CPPS animal model, the change in pathology of the animal’s prostate tissue is most similar to that of patients, and pathogenesis is relatively consistent with patients. It is a relatively satisfied way to induce CPPS animal model.②Qianlietongyu-tang can reduce or eliminate inflammation of the model rat prostate tissue, improve its organizational structure damage, it has an anti-fibrosis effect, and so plays a significant role in the protection of prostate issue.③Qianlietongyu-tang can dramatically reduce the levels of IL-1β, COX-2, PGE2 of prostate tissue of the model rat, which can reduce inflammation reaction and pain of reproductive pelvic. The content ofβ-endorphin was dramatically increased in the high dose Qianlietongyu-tang group and celecoxib group, which can strengthen the effectiveness of topo-inhibition pain. Those two groups showed no significant difference in all parameters.
The research demonstrated that the possible mechanism of treatment of Qianlietongyu Tang on CPPS is closely related to the effect of adjusting the prostate tissue cytokine levels, reduce prostate inflammation and protect prostate tissue.
研究显示,前列腺素E2(Prostaglandin-E2,PGE2)在CPPS患者的前列腺液(expressed prostatic secretions, EPS)中明显升高,是引起疼痛的主要原因。组织损伤、炎症和局部感染等使白细胞介素-1β(interleukin-1β,IL-1β)产生增多,继而诱导环氧化酶-2(Cyclooxygenase-2,COX-2)的基因表达,COX-2的产生迅速增多,促使细胞产生大量的PGE2,高浓度的PGE2又能抑制β-內啡肽(β-endorphin,β-EP)的分泌和调节疼痛的功能。应用特异性COX-2抑制剂治疗CPPS可明显减轻疼痛。我们推测前列通瘀汤可能是通过降低PGE2而对CPPS产生治疗作用。本课题旨在通过动物实验研究,探讨前列通瘀汤对PGE2等细胞因子的干预作用,揭示其治疗CPPS的可能机制。
目的本课题采用免疫佐剂法制作实验性CPPS大鼠模型,观察前列通瘀汤对实验大鼠前列腺组织形态学的影响,评价其疗效;以COX-2抑制剂塞来昔布为阳性对照药,观察前列通瘀汤对致痛因子PGE2,及与之密切相关的IL-1β、COX-2和β-EP的影响,探讨前列通瘀汤治疗CPPS的可能机理,并总结其组方规律和用药特点,为指导临床合理选择有效的中药及方剂提供思路。
方法取3月龄180-220g雄性Wistar种大鼠62只,随机分为正常组10只、实验组52只。实验组大鼠采用同种系大鼠前列腺蛋白提纯液,辅以双重免疫佐剂方法,复制实验性CPPS大鼠模型。模型成功后,再随机将实验组大鼠分为模型组、塞来昔布组、前列通瘀汤大、中、小剂量组五组。前列通瘀汤大、中、小剂量组给药剂量分别为32、16、8g生药/kg。塞来昔布组给药剂量为0.04 g/kg。正常组和模型组给于相同剂量的蒸馏水。以上各组均灌胃给药,每日一次,连续给药30d。第30天将各组大鼠称重后脱颈处死。取前列腺组织,观察其病理改变用ELISA法测定前列腺组织匀浆IL-1β、COX-2、PGE2、β-EP的含量。
结果①病理观察显示,模型组大鼠前列腺组织表现为慢性炎症的病理变化。其前列腺腺体普遍与周围组织粘连,腺体呈暗红色或棕黄色,质稍硬,或有明显的硬结。显微镜下主要表现为淋巴细胞和单核细胞的浸润,间质水肿及纤维组织增生,前列腺上皮破坏、萎缩及脱落,前列腺管壁的增生或破坏。对前列腺病理切片的分级进行统计分析,显示模型组大鼠与正常大鼠前列腺组织病理表现差异有统计学意义。表明造模成功。②前列通瘀汤大剂量组和塞来昔布组大鼠前列腺上皮组织恢复正常,管壁结构完整,间质水肿消失。前列通瘀汤中剂量组和小剂量组对实验大鼠前列腺组织结构的破坏虽然也有一定程度的改善,但作用较轻。病理分级统计结果显示:前列通瘀汤大、中、小剂量组、塞来昔布组,与模型组比较差异均有统计学意义;前列通瘀汤小剂量组与正常组比较差异有统计学意义,其余各组与正常组比较,差异无统计学意义。③前列通瘀汤大、中、小剂量组,及塞来昔布组大鼠前列腺组织内PGE2及IL-1β、COX-2的水平较模型组明显降低;β-EP的水平较模型组明显升高。其中前列通瘀汤大剂量组与塞来昔布对照组比较,差异无统计学意义。
结论①采用自身免疫反应所造成的CPPS动物模型,其病理改变与临床的病理改变最为接近,发病机理也与人的发病较为符合,是一种较为理想的的造模方式。②前列通瘀汤能够减轻或消除模型大鼠前列腺组织的炎症反应,改善其组织结构的破坏,抗纤维化,对前列腺组织起到明显保护作用。③前列通瘀汤能够降低模型大鼠前列腺组织内PGE2及IL-1β、COX-2的水平,减轻前列腺组织炎症反应的程度,减轻盆腔生殖区域的疼痛;并能升高β-EP的水平,使局部抑制疼痛的作用加强。
研究表明,前列通瘀汤对CPPS的治疗作用可能与前列通瘀汤能调节前列腺组织的细胞因子含量,减轻前列腺组织炎症反应,对前列腺组织起到保护作用密切相关。
Chronic non-bacterial prostatitis/chronic pelvic pain syndrome (CPPS) is a common disease of male reproductive system, mainly to the reproductive pelvic pain and abnormal urination.The etiology and the pathogenesis of CPPS are not yet clear by now. Because of the high incidence of the disease, lack of typical symptoms, and difficult to treat, CPPS seriously impacts on the quality of life in patients. Recently studies have shown that CPPS is the result of a variety of factors working together, and autoimmune reactions play an important role in the pathogenesis, which is mediated by inflammatory cytokines. Traditional Chinese Medicine has some advantages in the treatment of chronic prostatitis by treatment based on syndrome differentiation, and already became one of the important ways of the clinical treatment. Qianlietongyu-tang is a Chinese medicine that can promote blood flow, relieve fever and promote urination. It can achieve a significant effect in treating CPPS, especially to relieve pain.
Research shows that prostaglandin E2 (PGE2) in CPPS prostatic fluid was significantly increased, and is the main cause of pain. Tissue injury, inflammation, and local infection, etc can cause increased production of interleukin-1 (IL-1). Increased IL-1 can induce the expression of cyclooxygenase-2 (COX-2) gene, resulting in a rapidly increase of COX-2, which induces a high production of PGE2 in the cells。High concentrations of PGE2 can inhibit the secretion and the analgesic effect ofβ-endorphin (β-EP). Specific COX-2 inhibitors can significantly reduce the pain of CPPS. We have conjectured that the possible treatment mechanism of Qianlietongyu-tang may be achieved by the way of decreasing the concentration of PGE2 of the prostate tissue. The topic made use of animal experiments to observe the intervention of Qianlietongyu-tang on cytokine such as PGE2, etc, and revealed its possible mechanism of treatment of CPPS.
Object The project made the experimental CPPS rat model by using SC purified prostate protein twice with immune adjuvant, observed the influence of Qianlietongyu-tang on morphological peculiarities of experimental rats prostate tissue, and evaluated its efficacy. The project observed the change of pain factor—PGE2, and the other cytokines as IL-1, COX-2 andβ2-EP that were closely related with PGE2, to explore the possible mechanism of treatment by Qianlietongyu-tang. The project also summarized the patterns of prescription and the characteristics of the drug to provide ideas for guiding reasonable choice of effective medicines and prescriptions in clinic.
Methods Selection of 62 male, 3-month-old, weight 180-220g Wistar rats, randomly divided into two groups: a control group, 10 rats and a model group, 52 rats. The model group was made by DPT vaccine in peritoneal injection, and rat prostatein purification of FCA emulsion multiple intra-dermal injections. The injected rats were again randomly divided into a control group, a model group, a celecoxib group and a group of low, middle and high dose of Qianlietongyu-tang. The low, middle and high dose group was given dose according to standards of 32,16,8g crude drug/kg respectively. The Celecoxib group administrates the dose of 0.04/kg. The control group and the model group were given the same dose of distilled water. The above groups were successively administrated oral for 30days, qd. All animals were killed 30d after treatment, and prostate glands were obtained to observe the histomorphological changes and detect the contents of IL-1β, COX-2, PGE2 andβ-endorphin in prostate-homogenate by enzyme linked immunosorbent assay.
Results①pathological findings: the model group shows chronic inflammation of prostate glandular tissue. The glandular organ of prostate gland adheres to the surrounding tissue, kermesinus or yellow, slightly hard in quality, or obviously indurate. Under microscope, infiltration of lymph and mononuclear cells, interstitial edema and fibroplasias, damage, atrophia, or ablate of prostate gland endepidermis, accrementition or damage of prostatid ducts can be seen. According to statistical analysis on classified pathological sections of prostate gland, the difference between model group and normal group has statistical significance. The above results indicate that the model building is successful.②In the high dose group of Qianlietongyu-tang and Celecoxib group, the epithelial tissues recover, the architecture of epithelial tissues are integrated, and the interstitial edemas disappear. In the mid and low dose group, the damage of prostate gland tissues recover slightly. According to classified statistical analysis, the differences among each group of the Qianlietongyu-tang and the Celecoxib group had statistical significance. The difference between the low dose group of Qianlietongyu-tang and normal group had statistical significance, whereas the differences among the other groups had not.③Compared to the model group, the level of PGE2, IL-1βand COX-2 of prostate glandular tissues reduced in each group of Qianlietongyu-tang and Celecoxib, whereasβ-EP increased obviously. The difference between high dose group of Qianlietongyu-tang and Celecoxib group had no statistical significance.
Conclusion①Using the way of autoimmune reaction to induce the CPPS animal model, the change in pathology of the animal’s prostate tissue is most similar to that of patients, and pathogenesis is relatively consistent with patients. It is a relatively satisfied way to induce CPPS animal model.②Qianlietongyu-tang can reduce or eliminate inflammation of the model rat prostate tissue, improve its organizational structure damage, it has an anti-fibrosis effect, and so plays a significant role in the protection of prostate issue.③Qianlietongyu-tang can dramatically reduce the levels of IL-1β, COX-2, PGE2 of prostate tissue of the model rat, which can reduce inflammation reaction and pain of reproductive pelvic. The content ofβ-endorphin was dramatically increased in the high dose Qianlietongyu-tang group and celecoxib group, which can strengthen the effectiveness of topo-inhibition pain. Those two groups showed no significant difference in all parameters.
The research demonstrated that the possible mechanism of treatment of Qianlietongyu Tang on CPPS is closely related to the effect of adjusting the prostate tissue cytokine levels, reduce prostate inflammation and protect prostate tissue.
引文
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