初级传入神经元TRPM8在大鼠慢性关节炎性疼痛中的作用
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摘要
慢性疼痛的治疗一直是临床的难点,冷刺激可以缓解慢性疼痛并可避免常用镇痛药物的副作用,但过度的冷刺激却可诱发疼痛和冷觉过敏。TRPs通道是位于细胞膜上的一类重要的阳离子通道,在慢性疼痛中发挥重要作用,是近年研究的热点。TRPM8是TRPs通道家族的重要成员,主要感知冷刺激,可被包括薄荷醇、icilin等在内多种凉性化合物及低温(<28℃)激活。TRPM8的确定为研究冷刺激与疼痛的关系提供了新的途径和手段。本研究以建立关节炎大鼠模型为基础,通过薄荷醇及TRPM8反义寡核苷酸激活或者抑制TRPM8,着重探讨TRPM8在慢性炎性疼痛大鼠中的作用。期望这些研究结果能为日后相关炎性疼痛研究及治疗提供依据。
第一部分：关节炎大鼠背根神经节TRPM8表达的变化
目的：建立完全弗氏佐剂关节炎大鼠,观察其背根神经节TRPM8的表达变化,以探讨TRPM8在冷觉过敏形成中的意义。方法：将30只健康雄性SD大鼠,体重300±20g,随机分成5组(n=6),制成弗氏佐剂关节炎模型,分别为关节炎1天组(CFA-1组)、关节炎4天组(CFA-4组)、关节炎7天组(CFA-7组)、关节炎10天组(CFA-10组)、关节炎14天组(CFA－14组)；将30只健康雄性SD大鼠随机分成5组(n=6),制成对照模型,分别为对照1天组(NS-1组)、对照4天组(NS-4组)、对照7天组(NS-7组)、对照10天组(NS-10组)、对照14天组(NS-14组)。动态监测大鼠行为学指标,以免疫组织化学方法检测大鼠DRG的TRPM8表达。结果：模型建立后随着时间的推移,大鼠右侧L5DRG的TRPM8表达上调,CFA-7组、CFA－10组、CFA-14组三组大鼠TRPM8表达水平达到峰值。对照组DRG的TRPM8表达没有变化。结论：DRG内TRPM8可能参与冷觉过敏的形成机制。
第二部分：薄荷醇和TRPM8反义寡核苷酸对慢性关节炎大鼠痛觉过敏和背根神经节TRPM8表达的影响
目的：建立完全弗氏佐剂关节炎大鼠模型,观察薄荷醇、反义寡核苷酸对大鼠行为学的影响以及背根神经节TRPM8的表达变化,以探讨TRPM8在慢性炎性痛大鼠中的作用。方法：健康雄性SD大鼠,体重300±20g,行鞘内置管,随机分成8组(n=8),其中4组制成弗氏佐剂关节炎模型,鞘内分别给予薄荷醇100μg/kg(CFA-Menthol组)、生理盐水20μl(CFA-NS组)、反义寡核苷酸60μg/kg(CFA-Antisense组)、错义寡核苷酸60μg/kg(CFA-Missense组)；另外4组制成对照组(假炎症组),鞘内分别给予薄荷醇100μg/kg(NS-Menthol组)、生理盐水20μl(NS-NS)组)、反义寡核营酸60μg/kg(NS-Antisense组)、错义寡核苷酸60μg/kg(NS-Missense组)。其中,CFA-Menthol组、CFA-NS组、NS-Menthol组、NS-NS组大鼠致炎后第14天给药1次；CFA-Antisense组、CFA-Missense组、NS-Antisense组、NS-Missense组致炎后第9天给药,每天2次,连续5天。动态检测大鼠冷板指标、机械缩爪阈值及热板缩爪潜伏期。采用免疫组化、western blot检测以上各组右侧L5DRG内TRPM8的表达。结果：CFA-Menthol组大鼠鞘内给予薄荷醇后冷觉过敏增强,热痛敏和机械痛敏有所逆转；CFA-Antisense组大鼠冷觉过敏有所缓解。免疫组化及western结果显示,CFA-Menthol组、CFA-NS组以及CFA-Missense组致炎大鼠右L5DRG内TRPM8表达上调,鞘内给予反义寡核苷酸的CFA-Antisense组、NS-Antisense组大鼠右L5DRG内TRPM8表达下调。结论：鞘内给予薄荷醇激活表达上调的TRPM8使致炎大鼠冷觉过敏增强,热、机械痛敏逆转；鞘内给予反义寡核苷酸阻断TRPM8表达,使致炎大鼠冷觉过敏有所缓解,而对非致炎大鼠的行为学没有影响。慢性炎性疼痛大鼠的冷觉过敏的变化与TRPM8表达变化有关,薄荷醇的镇痛作用与TRPM8表达上调有关。
第三部分：薄荷醇、TRPM8反义寡核苷酸的ED50、ED95的测定
目的：建立完全弗氏佐剂关节炎大鼠模型,测量大鼠对薄荷醇、反义寡核苷酸反应的ED50、ED95,以探讨其在炎性痛大鼠中的量效关系。方法：健康雄性SD大鼠,体重300±20g,行鞘内置管,随机分为14组(n=10),制成弗氏佐剂关节炎模型,其中7组第14天鞘内分别给予生理盐水20μl、薄荷醇31.62μg/kg、39.81μg／kg、50.12μg／kg、63.10μg／kg、79.43μg／kg、100.00μg／kg；另7组致炎后第9天鞘内分别给予生理盐水20μl、反义寡核苷酸18.97gg／kg、23.89gg／kg、30.07μg／kg、37.86μg／kg、47.66μg／kg、60.00μg／kg,每天2次,连续5天；第14天冷板测量大鼠右后足抬足次数,以盐水对照组抬足次数为标准,薄荷醇组升高30%记为有效,反义寡核苷酸组降低30%记为有效。按Probit模型分别计算薄荷醇、反义寡核苷酸的ED50及ED95。结果：薄荷醇的ED50、ED95分别为：53.36μg／kg、88.31μg／kg；反义寡核苷酸的ED50、ED95分别为：29.92μg／kg、50.36μg／kg。结论：薄荷醇的ED50、ED95分别为：53.36μg／kg、88.31μg／kg；反义寡核苷酸的ED50、ED95分别为：29.92μg／kg、50.36μg／kg。
The treatment of chronic pain has been the difficulty in clinical. Cold stimulus can alleviate chronic pain and avoid the side effects of commonly used analgesic drugs. However, excessive cold can evoke pain and induce hypercryalgesia. Transient receptor potential channels(TRPs), the research hotspot in recent years, which plays an important role in chronic pain, is an important kind of cation channel located on cell membrane. TRPM8, a member of TRP channel family, is activated by cool compounds such as menthol, icilin, and by temperature below 28℃.The identification of TRPM8 produces profound, novel methods to relieve pain. In the dissertation, a model of chronic arthritic rats was constructed. Based on this model, the effect of TRPM8 on the chronic arthritic rats was explored when it was activated by menthol or blocked by the antisense oligonucleotides.The results of this study are expected to provide important information for future treatment and studies associated with chronic arthritic pain.
Part one:Alteration of TRPM8 expression in dorsal root ganglion of arthritic rats.
Objective:To establish the rat model of complete Freund's adjuvant-induced arthritis, observe the changes of TRPM8 expression in dorsal root ganglion of the arthritic rats, and analyze the significance of TRPM8 on the development of hypercryalgesia. Methods:The 30 healthy male SD rats, weighed 300±20g, which were made into model of adjuvant induced arthritis, were randomized into 5 groups(n=6), as 1 day arthritis group (group CFA-1),4 day arthritis group (group CFA-4),7 day arthritis group (group CFA-7),10 day arthritis group (group CFA-10),14 day arthritis group (group CFA-14).The other 30 healthy male SD rats were made into control model without arthritis, randomized into 5 groups(n=6), as 1 day in control group (group NS-1),4 day in control group (group NS-4),7 day in control group (group NS-7),10 day in control group (group NS-10),14 day in control group (group NS-14). Behavioral indicators of rats were examined every day. Immunohistochemistry was used to detect the expression of TRPM8 in dorsal root ganglion. Results:As model established, the expression of TRPM8 was up-regulation which reached a peak level in groups CFA-7, CFA-10 and CFA-14, while the expression of TRPM8 in the control groups was not changed. Conclusion:TRPM8 expressed in DRG may be involved in the mechanism of cold allergy in arthritic rats.
Part two:Effects of menthol and TRPM8-antisense oligonucleotides on hyperalgesia and the experssion of TRPM8 in dorsal root ganglion of rats with chronic arthritis
Objective:To establish the rat model of complete Freund's adjuvant-induced arthritis, observe the effect of menthol and TRPM8-antisense oligonucleotides on the behavioral indicators and the expression of TRPM8 in dorsal root ganglion of chronic arthritic rats, and analyze the significance of TRPM8 in chronic inflammatory pain rats. Methods:Healthy male SD rats, weighed 300±20g, were implanted intrathecal catheters and randomized into 8 groups. Amongst, the rats in 4 groups were made into model of adjuvant-induced arthritis and administered intrathecally with menthol 100μg/kg(group CFA-Menthol), saline 20μ(group CFA-NS), antisense oligonucleotid-es 60μg/kg(group CFA-Antisense), missense oligonucleotides 60μg/kg(group CFA-Missense). The rest rats in the other 4 groups were made into control model without athritis and administered intrathecally with menthol 100μg/kg (group NS-Menthol), saline 20μl(group NS-NS), antisense oligonucleotides 60μg/kg(group NS-Antisense), missense oligonucleotides 60μg/kg(group NS-Missense). Drugs were given once in the 14th day in group CFA-Menthol, CFA-NS, NS-Menthol and NS-NS, while given twice a day for five days from the 9th day in the rest groups.The cold plate indicators, mechanical withdrawal threshold (MWT) and thermal paw withdrawal latency (PWL) of rats was examined to evaluate their behavior. Immunohistochemical stain and western blot were used to measure the expression of TRPM8 in the rats'right L5 dorsal root ganglion. Results:After applied menthol, the hypercryalgesia of rats in group CFA-Menthol was enhanced, while the hyperthermalgesia and the mechanical pain were reversed. And the hypercryalgesia of rats in group CFA-Antisense was inhibited. The expression of TRPM8 was up-regulation in ipsilateral L5 dorsal root ganglion of the rats in group CFA-Menthol, CFA-NS and CFA-Missense with athritis,while blocked in goup CFA-Antisense and NS-Antisense after applied with TRPM8-antisense oligonucleotides. Conclusion:TRPM8 in athritic rats, activated by menthol given intrathecally, enhanced the hypercryalgesia while reversed the hyperthermalgesia and the mechanical pain.TRPM8, blocked by TRPM8-antisense oligonucleotides, inhibited the hypercryalgesia in athritic rats while had no effect on normal rats.The changes of chronic arthritic rats'hypercryalgesia were concerned with alteration of TRPM8 expression and the analgesic effect of menthol was related with the up-regulation of TRPM8.
Part three:Measurement of the ED50 and ED95 of intrathecal menthol and TRPM8-antisense oligonucleotides for arthritic rats.
Objective:To establish the rat model of complete Freund's adjuvant-induced arthritis, measure the the ED50 and ED95 of intrathecal menthol and TRPM8-antisense oligonucleotides for arthritic rats, and analyze the dose-effect relationship of menthol and TRPM8-antisense oligonucleotides in chronic arthritic rats. Methods: 140 healthy male SD rats, weighed 300±20g, were implanted intrathecal catheters, made into model of adjuvant-induced artliritis and randomized into 14 groups(n=10). Amongst, the rats in 7 groups were applied intrathecally saline 20μl, menthol 31.62μg/kg,39.81μg/kg,50.12μg/kg,63.10μg/kg,79.43μg/kg, 100.00μg/kg in the fourteenth day. The rats in the other 7 groups were applied intrathecally saline 20μl, antisense oligonucleotides 18.97μg/kg,23.89μg/kg,30.07μg/kg,37.86μg/kg,47.66μg/kg,60.00μg/kg, twice a day for five days from the ninth day.The number of the right hind paw lifts was measured. And if the number in group menthol or antisense oligonucleotides was thirty percent higher or lower than that of group NS, it would be confirmed as effective. ED50 and ED95 values were calculated using a Probit Regression models. Results:ED50 and ED95 values of menthol were 53.36μg/kg, 88.31μg/kg. ED50 and ED95 values of antisense oligonucleotides were 29.92μg/kg, 50.36μg/kg.Conclusion:ED50 and ED95 values of menthol were 53.36μg/kg, 88.31μg/kg.ED50 and ED95 values of antisense oligonucleotides were 29.92μg/kg, 50.36μg/kg.
第一部分：关节炎大鼠背根神经节TRPM8表达的变化
目的：建立完全弗氏佐剂关节炎大鼠,观察其背根神经节TRPM8的表达变化,以探讨TRPM8在冷觉过敏形成中的意义。方法：将30只健康雄性SD大鼠,体重300±20g,随机分成5组(n=6),制成弗氏佐剂关节炎模型,分别为关节炎1天组(CFA-1组)、关节炎4天组(CFA-4组)、关节炎7天组(CFA-7组)、关节炎10天组(CFA-10组)、关节炎14天组(CFA－14组)；将30只健康雄性SD大鼠随机分成5组(n=6),制成对照模型,分别为对照1天组(NS-1组)、对照4天组(NS-4组)、对照7天组(NS-7组)、对照10天组(NS-10组)、对照14天组(NS-14组)。动态监测大鼠行为学指标,以免疫组织化学方法检测大鼠DRG的TRPM8表达。结果：模型建立后随着时间的推移,大鼠右侧L5DRG的TRPM8表达上调,CFA-7组、CFA－10组、CFA-14组三组大鼠TRPM8表达水平达到峰值。对照组DRG的TRPM8表达没有变化。结论：DRG内TRPM8可能参与冷觉过敏的形成机制。
第二部分：薄荷醇和TRPM8反义寡核苷酸对慢性关节炎大鼠痛觉过敏和背根神经节TRPM8表达的影响
目的：建立完全弗氏佐剂关节炎大鼠模型,观察薄荷醇、反义寡核苷酸对大鼠行为学的影响以及背根神经节TRPM8的表达变化,以探讨TRPM8在慢性炎性痛大鼠中的作用。方法：健康雄性SD大鼠,体重300±20g,行鞘内置管,随机分成8组(n=8),其中4组制成弗氏佐剂关节炎模型,鞘内分别给予薄荷醇100μg/kg(CFA-Menthol组)、生理盐水20μl(CFA-NS组)、反义寡核苷酸60μg/kg(CFA-Antisense组)、错义寡核苷酸60μg/kg(CFA-Missense组)；另外4组制成对照组(假炎症组),鞘内分别给予薄荷醇100μg/kg(NS-Menthol组)、生理盐水20μl(NS-NS)组)、反义寡核营酸60μg/kg(NS-Antisense组)、错义寡核苷酸60μg/kg(NS-Missense组)。其中,CFA-Menthol组、CFA-NS组、NS-Menthol组、NS-NS组大鼠致炎后第14天给药1次；CFA-Antisense组、CFA-Missense组、NS-Antisense组、NS-Missense组致炎后第9天给药,每天2次,连续5天。动态检测大鼠冷板指标、机械缩爪阈值及热板缩爪潜伏期。采用免疫组化、western blot检测以上各组右侧L5DRG内TRPM8的表达。结果：CFA-Menthol组大鼠鞘内给予薄荷醇后冷觉过敏增强,热痛敏和机械痛敏有所逆转；CFA-Antisense组大鼠冷觉过敏有所缓解。免疫组化及western结果显示,CFA-Menthol组、CFA-NS组以及CFA-Missense组致炎大鼠右L5DRG内TRPM8表达上调,鞘内给予反义寡核苷酸的CFA-Antisense组、NS-Antisense组大鼠右L5DRG内TRPM8表达下调。结论：鞘内给予薄荷醇激活表达上调的TRPM8使致炎大鼠冷觉过敏增强,热、机械痛敏逆转；鞘内给予反义寡核苷酸阻断TRPM8表达,使致炎大鼠冷觉过敏有所缓解,而对非致炎大鼠的行为学没有影响。慢性炎性疼痛大鼠的冷觉过敏的变化与TRPM8表达变化有关,薄荷醇的镇痛作用与TRPM8表达上调有关。
第三部分：薄荷醇、TRPM8反义寡核苷酸的ED50、ED95的测定
目的：建立完全弗氏佐剂关节炎大鼠模型,测量大鼠对薄荷醇、反义寡核苷酸反应的ED50、ED95,以探讨其在炎性痛大鼠中的量效关系。方法：健康雄性SD大鼠,体重300±20g,行鞘内置管,随机分为14组(n=10),制成弗氏佐剂关节炎模型,其中7组第14天鞘内分别给予生理盐水20μl、薄荷醇31.62μg/kg、39.81μg／kg、50.12μg／kg、63.10μg／kg、79.43μg／kg、100.00μg／kg；另7组致炎后第9天鞘内分别给予生理盐水20μl、反义寡核苷酸18.97gg／kg、23.89gg／kg、30.07μg／kg、37.86μg／kg、47.66μg／kg、60.00μg／kg,每天2次,连续5天；第14天冷板测量大鼠右后足抬足次数,以盐水对照组抬足次数为标准,薄荷醇组升高30%记为有效,反义寡核苷酸组降低30%记为有效。按Probit模型分别计算薄荷醇、反义寡核苷酸的ED50及ED95。结果：薄荷醇的ED50、ED95分别为：53.36μg／kg、88.31μg／kg；反义寡核苷酸的ED50、ED95分别为：29.92μg／kg、50.36μg／kg。结论：薄荷醇的ED50、ED95分别为：53.36μg／kg、88.31μg／kg；反义寡核苷酸的ED50、ED95分别为：29.92μg／kg、50.36μg／kg。
The treatment of chronic pain has been the difficulty in clinical. Cold stimulus can alleviate chronic pain and avoid the side effects of commonly used analgesic drugs. However, excessive cold can evoke pain and induce hypercryalgesia. Transient receptor potential channels(TRPs), the research hotspot in recent years, which plays an important role in chronic pain, is an important kind of cation channel located on cell membrane. TRPM8, a member of TRP channel family, is activated by cool compounds such as menthol, icilin, and by temperature below 28℃.The identification of TRPM8 produces profound, novel methods to relieve pain. In the dissertation, a model of chronic arthritic rats was constructed. Based on this model, the effect of TRPM8 on the chronic arthritic rats was explored when it was activated by menthol or blocked by the antisense oligonucleotides.The results of this study are expected to provide important information for future treatment and studies associated with chronic arthritic pain.
Part one:Alteration of TRPM8 expression in dorsal root ganglion of arthritic rats.
Objective:To establish the rat model of complete Freund's adjuvant-induced arthritis, observe the changes of TRPM8 expression in dorsal root ganglion of the arthritic rats, and analyze the significance of TRPM8 on the development of hypercryalgesia. Methods:The 30 healthy male SD rats, weighed 300±20g, which were made into model of adjuvant induced arthritis, were randomized into 5 groups(n=6), as 1 day arthritis group (group CFA-1),4 day arthritis group (group CFA-4),7 day arthritis group (group CFA-7),10 day arthritis group (group CFA-10),14 day arthritis group (group CFA-14).The other 30 healthy male SD rats were made into control model without arthritis, randomized into 5 groups(n=6), as 1 day in control group (group NS-1),4 day in control group (group NS-4),7 day in control group (group NS-7),10 day in control group (group NS-10),14 day in control group (group NS-14). Behavioral indicators of rats were examined every day. Immunohistochemistry was used to detect the expression of TRPM8 in dorsal root ganglion. Results:As model established, the expression of TRPM8 was up-regulation which reached a peak level in groups CFA-7, CFA-10 and CFA-14, while the expression of TRPM8 in the control groups was not changed. Conclusion:TRPM8 expressed in DRG may be involved in the mechanism of cold allergy in arthritic rats.
Part two:Effects of menthol and TRPM8-antisense oligonucleotides on hyperalgesia and the experssion of TRPM8 in dorsal root ganglion of rats with chronic arthritis
Objective:To establish the rat model of complete Freund's adjuvant-induced arthritis, observe the effect of menthol and TRPM8-antisense oligonucleotides on the behavioral indicators and the expression of TRPM8 in dorsal root ganglion of chronic arthritic rats, and analyze the significance of TRPM8 in chronic inflammatory pain rats. Methods:Healthy male SD rats, weighed 300±20g, were implanted intrathecal catheters and randomized into 8 groups. Amongst, the rats in 4 groups were made into model of adjuvant-induced arthritis and administered intrathecally with menthol 100μg/kg(group CFA-Menthol), saline 20μ(group CFA-NS), antisense oligonucleotid-es 60μg/kg(group CFA-Antisense), missense oligonucleotides 60μg/kg(group CFA-Missense). The rest rats in the other 4 groups were made into control model without athritis and administered intrathecally with menthol 100μg/kg (group NS-Menthol), saline 20μl(group NS-NS), antisense oligonucleotides 60μg/kg(group NS-Antisense), missense oligonucleotides 60μg/kg(group NS-Missense). Drugs were given once in the 14th day in group CFA-Menthol, CFA-NS, NS-Menthol and NS-NS, while given twice a day for five days from the 9th day in the rest groups.The cold plate indicators, mechanical withdrawal threshold (MWT) and thermal paw withdrawal latency (PWL) of rats was examined to evaluate their behavior. Immunohistochemical stain and western blot were used to measure the expression of TRPM8 in the rats'right L5 dorsal root ganglion. Results:After applied menthol, the hypercryalgesia of rats in group CFA-Menthol was enhanced, while the hyperthermalgesia and the mechanical pain were reversed. And the hypercryalgesia of rats in group CFA-Antisense was inhibited. The expression of TRPM8 was up-regulation in ipsilateral L5 dorsal root ganglion of the rats in group CFA-Menthol, CFA-NS and CFA-Missense with athritis,while blocked in goup CFA-Antisense and NS-Antisense after applied with TRPM8-antisense oligonucleotides. Conclusion:TRPM8 in athritic rats, activated by menthol given intrathecally, enhanced the hypercryalgesia while reversed the hyperthermalgesia and the mechanical pain.TRPM8, blocked by TRPM8-antisense oligonucleotides, inhibited the hypercryalgesia in athritic rats while had no effect on normal rats.The changes of chronic arthritic rats'hypercryalgesia were concerned with alteration of TRPM8 expression and the analgesic effect of menthol was related with the up-regulation of TRPM8.
Part three:Measurement of the ED50 and ED95 of intrathecal menthol and TRPM8-antisense oligonucleotides for arthritic rats.
Objective:To establish the rat model of complete Freund's adjuvant-induced arthritis, measure the the ED50 and ED95 of intrathecal menthol and TRPM8-antisense oligonucleotides for arthritic rats, and analyze the dose-effect relationship of menthol and TRPM8-antisense oligonucleotides in chronic arthritic rats. Methods: 140 healthy male SD rats, weighed 300±20g, were implanted intrathecal catheters, made into model of adjuvant-induced artliritis and randomized into 14 groups(n=10). Amongst, the rats in 7 groups were applied intrathecally saline 20μl, menthol 31.62μg/kg,39.81μg/kg,50.12μg/kg,63.10μg/kg,79.43μg/kg, 100.00μg/kg in the fourteenth day. The rats in the other 7 groups were applied intrathecally saline 20μl, antisense oligonucleotides 18.97μg/kg,23.89μg/kg,30.07μg/kg,37.86μg/kg,47.66μg/kg,60.00μg/kg, twice a day for five days from the ninth day.The number of the right hind paw lifts was measured. And if the number in group menthol or antisense oligonucleotides was thirty percent higher or lower than that of group NS, it would be confirmed as effective. ED50 and ED95 values were calculated using a Probit Regression models. Results:ED50 and ED95 values of menthol were 53.36μg/kg, 88.31μg/kg. ED50 and ED95 values of antisense oligonucleotides were 29.92μg/kg, 50.36μg/kg.Conclusion:ED50 and ED95 values of menthol were 53.36μg/kg, 88.31μg/kg.ED50 and ED95 values of antisense oligonucleotides were 29.92μg/kg, 50.36μg/kg.
引文
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