痛风康对急性痛风性关节炎的抗炎作用及对TLRs/MyD88通路的影响
摘要
背景
随着社会经济的发展,人们的饮食结构、生活方式发生很大变化,再者,世界范围内尤其是我国,人口的老龄化日趋明显,痛风的患病率呈现出逐步上升的趋势,对人们的健康构成了越来越大的威胁。急性痛风性关节炎患者因疼痛反复发作,在就诊的痛风患者中所占比例最大,如不积极治疗极易致残、致畸形,影响关节功能。急性痛风性关节炎的防治,已成为急待研究和解决的课题。中医药防治痛风由来已久,有着丰富的理论及许多有效方药,对临床有效的方药进行进一步的研究、开发具有重要意义。
痛风康是导师刘友章教授依据补肾活血、清热祛湿的治疗思想,结合临床,创制的纯中药复方。在临床应用中取得了很好的疗效。前期的临床观察已初步证实了痛风康治疗急性痛风性关节炎的疗效,既可以有效地改善痛风性关节炎患者的关节症状,又可以降低患者血尿酸；实验研究表明痛风康能明显改善急性痛风性关节炎模型大鼠的炎细胞浸润,明显减轻滑膜组织的损坏,改善血管扩张充血,具有明显的抗肿胀及镇痛的作用,但其作用机理需要进一步阐明。
近年来关于急性痛风的机理研究又有了新的发现：研究提示尿酸钠结晶能类似于外源性的佐剂一样作为危险信号起作用,Toll样受体(toll-like receptors,TLRs)能识别此类“危险信号”。髓样分化因子(myeloid differentiation factor 88,MyD88)是TLRs信号通路中的一个关键接头分子,在传递上游信息和疾病发生发展中具有重要的作用。MyD88依赖性信号转导途径是TLRs信号传导的共同通路。另外,尿酸钠(monosodium urate,MSU)结晶也引起吞噬细胞中髓样细胞触发受体1(triggering receptors expressed on myeloid cells 1,TREM-1)的上调,TLRs与TREM-1信号转导机制有关。激活TLRs可使TREM-1表达水平上升,TREM-1表达水平可改变TLR4信号转导途径关键受体和效应蛋白的表达,从而调节TLR4途径效应。因而,TREM-1与TLRs信号通路可能存在协同作用。观察痛风康对TLRs、MyD88、TREM-1的表达影响有助于进一步阐明其作用机制。
目的
观察痛风康治疗急性痛风性关节炎的临床疗效和安全性,观察痛风康对急性痛风性关节炎患者血清TREM-1的表达的影响；观察痛风康对急性痛风性关节炎大鼠关节症状及关节滑膜组织中TLR2、TLR4、MyD88表达的影响,从而进一步验证痛风康治疗急性痛风性关节炎的疗效并探讨其作用机理。
方法
临床研究：急性痛风性关节炎患者40例,随机分为治疗组和对照组,每组20例。治疗组采用痛风康颗粒和痛风外敷方治疗,对照组采用安慰剂治疗,疗程5天。记录患者关节疼痛、关节急性红肿、关节活动受限情况；患者记录自评疼痛得分。检测治疗前后患者肝肾功能。采用酶联免疫吸附(ELISA)法检测治疗前后患者血清TREM-1的表达。
实验研究：48只大鼠随机分为正常组、模型组、秋水仙碱组、痛风康高、中、低剂量组,每组8只。痛风康高、中、低剂量组的给药剂量分别为72g/kg/d、36g/kg/d、18g/kg/d,秋水仙碱组给药剂量为0.3mg/kg/d,正常组和模型组采用生理盐水灌胃,疗程5天。用药第3天,除正常组外所有大鼠均采用尿酸钠溶液关节腔注射方法造模,建立急性痛风性关节炎模型,正常组注入相同体积的生理盐水。动态测量大鼠右踝关节周径,观察大鼠炎症指数及功能障碍指数。HE染色法观察大鼠关节滑膜组织的病理变化。免疫组化法检测大鼠关节滑膜组织的TLR2、TLR4、MyD88的表达。
结果
临床研究：
(1)与对照组相比：痛风康治疗组第4天、第5天患者自评关节疼痛评分明显下降(第4天：P<0.05；第5天：P<0.01),第3天、第6天患者关节压痛减轻(均P<0.05),第3天、第6天关节急性红肿减轻(均P<0.05),第6天关节活动受限程度减轻(P<0.05),使用止痛药的情况较对照组减少(P<0.05)
(2)痛风康治疗组患者谷丙转氨酶、肌酐、尿素氮等安全性指标治疗前后均无明显变化(谷丙转氨酶：P>0.05；肌酐：P>0.05；尿素氮P>0.05)
(3)治疗后痛风康治疗组血清TREM-1浓度明显低于对照组(P<0.05)。痛风康治疗组血清TREM-1浓度下降幅度较对照组显著,对照组下降了28.9%,而治疗组下降了42.4%。
实验研究：
(1)与正常组相比,模型组造模后4、10、24、72小时关节肿胀均显著增加(4h：P<0.01；10h：P<0.01；24h：P<0.01；72h：P<0.01)。与模型组相比,秋水仙碱组造模后4、10、24、72小时关节肿胀均显著减轻(4h：P<0.05；10h：P<0.05；24h：P<0.05；72h：P<0.01)；痛风康高、中、低剂量组造模后4、10、24小时关节肿胀有减轻的趋势,72小时关节肿胀均显著减轻(痛风康高剂量组：P<0.01；痛风康中剂量组：P<0.01；痛风康低剂量组：P<0.01),与秋水仙碱组比较无统计学差异(痛风康高剂量组：P>0.05；痛风康中剂量组：P>0.05；痛风康低剂量组：P>0.05),痛风康高、中剂量组关节肿胀指数有较低剂量组降低的趋势。
模型组10h表现出明显的关节炎症红肿,到24h、72h表现出逐步缓解的趋势；与模型组比较,秋水仙碱组、痛风康低、中、高剂量组关节炎症红肿明显减轻,到72h关节仅表现轻度肿胀。
模型组10h表现出明显的关节功能障碍,到24h、72h逐步缓解；与模型组比较,秋水仙碱组、痛风康低、中、高剂量组关节功能障碍明显减轻,到72h关节功能障碍基本消失。
(2)HE染色显示模型大鼠关节滑膜炎症明显,滑膜细胞增生,伴炎细胞浸润,小血管增生及成纤维细胞,提示造模成功。经痛风康和秋水仙碱治疗后,上述病理改变减轻。
(3)正常组大鼠关节滑膜组织可见极少的TLR2表达。与正常组相比,模型组大鼠关节滑膜组织TLR2表达明显增多(P<0.01)。与模型组相比,秋水仙碱组滑膜组织TLR2表达明显减少(P<0.01),痛风康低、中、高剂量组滑膜组织TLR2表达明显减少(痛风康低剂量组：P<0.0)；痛风康中剂量组：P<0.01；痛风康高剂量组：P<0.01)痛风康低、中、高剂量组TLR2表达呈递减趋势。与秋水仙碱组比较,痛风康低、中、高剂量组滑膜组织TLR2表达无明显差别。
(4)正常组大鼠关节滑膜组织可见极少的TLR4表达。与正常组相比,模型组大鼠关节滑膜组织TLR4表达明显增多(P<0.01)。与模型组相比,秋水仙碱组滑膜组织TLR4表达明显减少(P<0.05),痛风康低、中、高剂量组滑膜组织TLR4表达明显减少(痛风康低剂量组：P<0.01,痛风康中剂量组：P<0.05,痛风康高剂量组：P<0.01)。与秋水仙碱组比,痛风康低、中、高剂量组滑膜组织TLR4表达无统计学差异。
(5)正常组大鼠关节滑膜组织可见较少的MyD88表达。与正常组相比,模型组大鼠关节滑膜组织MyD88表达明显增多(P<0.01)。与模型组相比,秋水仙碱组滑膜组织MyD88表达明显减少(P<0.01),痛风康低、中、高剂量组滑膜组织MyD88表达均减少(痛风康低剂量组：P<0.01,痛风康中剂量组：P<0.01,痛风康高剂量组：P<0.01),痛风康高、中剂量MyD88表达有较低剂量组进一步降低的趋势。与秋水仙碱组比,痛风康低、中、高剂量组滑膜组织MyD88表达无统计学差异。
结论
(1)痛风康可明显改善急性痛风性关节炎患者关节疼痛、红肿及功能障碍,且用药安全,对患者肝肾功能无明显影响。
(2)痛风康可明显改善MSU致急性痛风性关节炎模型大鼠的关节肿胀、红肿及功能障碍,改善滑膜组织炎症病理变化。
(3)痛风康可明显下调急性痛风性关节炎患者血清TREM-1的表达,对TREM-1表达的抑制可能是痛风康治疗急性痛风性关节炎的作用机制之一。
(4)痛风康可明显下调MSU致炎症关节滑膜组织的TLR2、TLR4、MyD88的表达,对TLRs/MyD88通路的干预,可能是痛风康治疗急性痛风性关节炎的作用机制之一。
Background
With the social and economic development, people's diet and lifestyle change greatly. Furthermore, aging of the population in the world has become more evident especially in China, so the prevalence of gout shows an increasing trend generally. It poses a threat to people's health. Because acute gouty arthritis leads to the repeated attack of pain, it will easily disabled, teratogenic, and affect joint function if not treated actively. Patients with acute gouty arthritis occupied the biggest percentage in visiting patients with gout. Prevention and treatment of acute gouty arthritis should be studied and solved urgently. Traditional Chinese Medicine(TCM) has been long for prevention and treatment of gout. TCM is rich in theory and effective herbs in the treatment of gout, so it is of great significance and prospects for further research and development of effective prescriptions.
According to therapeutic thoughts of reinforcing Kidney and activating blood, eliminating heat and wetness evil, Professor Liu Youzhang created the TCM compound formula named as TongFengKang(TFK) on the basis of long-term clinical practice. Previous clinical researchs have confirmed preliminary that effect of TFK on the acute gouty arthritis. TFK not only can improve patients'joint symptoms with acute gouty arthritis, but also can effectually decrease uric acid in patients'blood. Preliminary animal experiments have shown that TFK can reduce inflammatory cells infiltration, relieve the damage of synovial tissue, and reduce hyperemia in model rats with acute gouty arthritis. TFK have significant effect on swelling inhibition and analgesia, but its action mechanism needs to be further clarified.
Recent investigations provided novel evidence in the pathology of acute gout. A number of studies indicated that MSU crystals could act as a "danger signal" which resembled exogenous adjuvants, and toll-like receptors(TLRs) could recognize such "danger signals". Myeloid differentiation factor 88(MyD88) was a key adapter molecule in TLRs signaling pathway, which had an important role in passing the upstream signal and the development of disease. MyD88-dependent pathway was the common pathway of TLRs signals. In addition, TLRs were involved in triggering receptors expressed on myeloid cells 1 (TREM-1) pathways. Activation of TLRs could lead to up-regulation of TREM-1, and TREM-1 expression could change the expression of the key receptor and effector proteins in TLR4 pathway. Thus, TREM-1 and TLRs pathways might have a synergistic effect. Observing the effect of TFK on TLR2, TLR4, MyD88, TREM-1 will be beneficial to further reveal it action mechanism.
Objectives
Study therapeutic effect and security of TFK on treatment of acute gouty arthritis, and observe the effect of TFK on expression of patients'serum (TREM-1) with acute gouty arthritis; Research the effect of TFK on joint symptom and TRL2, TLR4, MyD88 in rats synovial tissues with acute gouty arthritis, then further confirm efficacy of TFK and reveal its action mechanism.
Methods
Clinical research:
40 patients with acute gouty arthritis were randomly divided into two groups:treatment group and control group,20 patients in each group. The treatment group was treated by "TFK granule" and "Tongfeng external application", the control group was treated with placebo. The treatment duration was 5 days. Recorded the index of the patients'joint pain, acute swelling and dysfunction. Recorded self-rating joint pain index. Detected patients'hepatic and renal function in pre and post treatment. Testing the expression of TREM-1 in patients'serum before and after the treatment by enzyme linked immunosorbent assay(ELISA). Experimental study:
48 rats were randomly divided into normal group, model group, colchicine group, large dose TFK group, medium dose TFK group, small dose TFK group,8 rats in each group. TFK large, medium and small dose group were given TFK separately in the dose of 72g/kg per day,36g/kg per day,18g/kg per day. Colchicine group was given colchicine solution in the dose of 0.3mg/kg per day. Saline water was administered to the normal group and model group. All rats had been oral administered by the drugs for 5 days. On the third day of drug, acute gouty arthritis model was set up via articular cavity MSU injection except the normal group, the same volume of saline was injected to the normal group. Right ankle joint circumference of rats was measured dynamically. The indexes of rats'inflammation and dysfunction were observved. The pathological changes of rats joint synovial tissue were detected by HE staining. The expression of TLR2, TLR4, MyD88 in rats synovium were measured by immunohistochemistry.
Results
Clinical research:
(1)Compared with control group, self-rating joint pain index of TFK treated group was significantly lightened(day4:P<0.05; day5:P<0.01), articular pain was lightened (day 3:P<0.05; day 6:P<0.05), acute swelling was lightened (day 3:P<0.05; day 6:P<0.05), dysfunction was lightened(day6:P<0.05), analgesic use decreased(P<0.05).
(2)Security indexes such as alanine aminotransferase(ALT), urea nitrogen (BUN), creatinine(CREA) of TFK treated group did not show significant change in pre and post treatment(ALT:P>0.05;BUN:P>0.05;CREA:P>0.05).
(3) Compared with control group, TREM-1 concentration of TFK treated group decreased significantly after treatment(P<0.05).The decrease before and after treatment of TFK treated group was more significant(decreased by 42.4%), whereas 28.9% in control group. Experimental study:
(1)Compared with normal group, the joint swelling index of model group was significantly increased(4h after model:P<0.01; 10h:P<0.01;24h:P<0.01; 72h: P<0.01). Compared with model group, the joint swelling index of colchicines group was significantly lightened(4h:P<0.05; 10h:P<0.05;24h:P<0.05; 72h: P<0.01); The joint swelling index of TFK large, medium and small dose group showed a decreasing trend at 4h, 10h and 24h, but was significantly lightened (TFK large dose group:P<0.01, TFK medium dose group:P<0.01 and TFK small dose group:P<0.01), but did not show significant change compared with colchicines group9 (TFK large dose group:P>0.05, TFK medium dose group:P>0.05 and TFK small dose group:P>0.05), and the joint swelling index of TFK large and medium dose group showed a decreasing trend compared with small dose group.
Model group showed significant joint inflammation(10h), and a gradually catabatic trend(24h,72h); Compared with model group, the joint inflammation of colchicines group, TFK small, medium and large dose group was significantly lightened, and was slight swelling at 72h.
Model group showed significant joint dysfunction(10h), and a catabatic gradually trend(24h,72h); Compared with model group, the joint dysfunction of colchicines group, TFK small, medium and large dose group was significantly lightened, and almost disappeared at 72h.
(2)The abnormalities of the rat's joint synovial tissue included obvious inflammation, synovial cells proliferation, inflammatory cell infiltration, small vessels proliferation, fibroblast cell by HE staining, which shown the animal model was successfully produced. After the treatment TFK and colchicines, these histological abnormalities were lessened.
(3)The TLR2 very few expression in joint synovial tissue of normal group. Compared with normal group, The TLR2 expression in joint synovial tissue of model group increased significantly(P<0.01). Compared with model group, The TLR2 expression in joint synovial tissue of colchicines group decreased significantly (P<0.01), The TLR2 expression in joint synovial tissue of TFK small, medium and large dose group decreased significantly(TFK small dose group:P<0.01; TFK medium dose group:P<0.01; TFK large dose group:P<0.01). TFK small, medium and large dose group showed a decreasing trend. Compared with colchicines group, The TLR2 expression in joint synovial tissue of TFK small, medium and large dose group did not show significant change.
(4)The TLR4 very few expression in joint synovial tissue of normal group. Compared with normal group, The TLR4 expression in joint synovial tissue of model group increased significantly(P<0.01). Compared with model group, The TLR4 expression in joint synovial tissue of colchicines group decreased significantly (P<0.05), The TLR2 expression in joint synovial tissue of TFK small, medium and large dose group decreased significantly(TFK small dose group:P<0.01; TFK medium dose group:P<0.05; TFK large dose group:P<0.01). Compared with colchicines group, The TLR4 expression in joint synovial tissue of TFK small, medium and large dose group did not show significant change.
(5)The MyD88 less expression in joint synovial tissue of normal group. Compared with normal group, The MyD88 expression in joint synovial tissue of model group increased significantly(P<0.01). Compared with model group, The MyD88 expression in joint synovial tissue of colchicines group decreased significantly(P<0.01), The MyD88 expression in joint synovial tissue of TFK small, medium and large dose group decreased significantly(TFK small dose group:P<0.01;TFK medium dose group:P<0.01; TFK large dose group: P<0.01). The MyD88 expression of TFK large and medium dose group showed a decreasing trend compared with small dose group. Compared with colchicines group, The TLR4 expression in joint synovial tissue of TFK small, medium and large dose group did not show significant change.
Conelusion
(1) TFK not only can significantly improve joint pain, swelling, dysfunctiong of acute gouty arthritis, but also was safe, there was no obvious impact on hepatic and renal function.
(2)TFK can significantly improve joint pain, swelling, dysfunctiong of rat with MSU-induced acute gouty arthritis.
(3)TFK can significantly reduce the expression of TREM-1 in patients'serum with acute gouty arthritis. The inhibition effect of TFK on the expression of TREM-1 was one of TFK mechanism in acute gouty arthritis treatment.
(4)TFK can significantly reduce the TLR2, TLR4 and MyD88 expression of synovium from inflammatory arthritis. The regulatory effect of TFK on TLRs/MyD88 pathways was one of TFK mechanism in acute gouty arthritis treatment.
随着社会经济的发展,人们的饮食结构、生活方式发生很大变化,再者,世界范围内尤其是我国,人口的老龄化日趋明显,痛风的患病率呈现出逐步上升的趋势,对人们的健康构成了越来越大的威胁。急性痛风性关节炎患者因疼痛反复发作,在就诊的痛风患者中所占比例最大,如不积极治疗极易致残、致畸形,影响关节功能。急性痛风性关节炎的防治,已成为急待研究和解决的课题。中医药防治痛风由来已久,有着丰富的理论及许多有效方药,对临床有效的方药进行进一步的研究、开发具有重要意义。
痛风康是导师刘友章教授依据补肾活血、清热祛湿的治疗思想,结合临床,创制的纯中药复方。在临床应用中取得了很好的疗效。前期的临床观察已初步证实了痛风康治疗急性痛风性关节炎的疗效,既可以有效地改善痛风性关节炎患者的关节症状,又可以降低患者血尿酸；实验研究表明痛风康能明显改善急性痛风性关节炎模型大鼠的炎细胞浸润,明显减轻滑膜组织的损坏,改善血管扩张充血,具有明显的抗肿胀及镇痛的作用,但其作用机理需要进一步阐明。
近年来关于急性痛风的机理研究又有了新的发现：研究提示尿酸钠结晶能类似于外源性的佐剂一样作为危险信号起作用,Toll样受体(toll-like receptors,TLRs)能识别此类“危险信号”。髓样分化因子(myeloid differentiation factor 88,MyD88)是TLRs信号通路中的一个关键接头分子,在传递上游信息和疾病发生发展中具有重要的作用。MyD88依赖性信号转导途径是TLRs信号传导的共同通路。另外,尿酸钠(monosodium urate,MSU)结晶也引起吞噬细胞中髓样细胞触发受体1(triggering receptors expressed on myeloid cells 1,TREM-1)的上调,TLRs与TREM-1信号转导机制有关。激活TLRs可使TREM-1表达水平上升,TREM-1表达水平可改变TLR4信号转导途径关键受体和效应蛋白的表达,从而调节TLR4途径效应。因而,TREM-1与TLRs信号通路可能存在协同作用。观察痛风康对TLRs、MyD88、TREM-1的表达影响有助于进一步阐明其作用机制。
目的
观察痛风康治疗急性痛风性关节炎的临床疗效和安全性,观察痛风康对急性痛风性关节炎患者血清TREM-1的表达的影响；观察痛风康对急性痛风性关节炎大鼠关节症状及关节滑膜组织中TLR2、TLR4、MyD88表达的影响,从而进一步验证痛风康治疗急性痛风性关节炎的疗效并探讨其作用机理。
方法
临床研究：急性痛风性关节炎患者40例,随机分为治疗组和对照组,每组20例。治疗组采用痛风康颗粒和痛风外敷方治疗,对照组采用安慰剂治疗,疗程5天。记录患者关节疼痛、关节急性红肿、关节活动受限情况；患者记录自评疼痛得分。检测治疗前后患者肝肾功能。采用酶联免疫吸附(ELISA)法检测治疗前后患者血清TREM-1的表达。
实验研究：48只大鼠随机分为正常组、模型组、秋水仙碱组、痛风康高、中、低剂量组,每组8只。痛风康高、中、低剂量组的给药剂量分别为72g/kg/d、36g/kg/d、18g/kg/d,秋水仙碱组给药剂量为0.3mg/kg/d,正常组和模型组采用生理盐水灌胃,疗程5天。用药第3天,除正常组外所有大鼠均采用尿酸钠溶液关节腔注射方法造模,建立急性痛风性关节炎模型,正常组注入相同体积的生理盐水。动态测量大鼠右踝关节周径,观察大鼠炎症指数及功能障碍指数。HE染色法观察大鼠关节滑膜组织的病理变化。免疫组化法检测大鼠关节滑膜组织的TLR2、TLR4、MyD88的表达。
结果
临床研究：
(1)与对照组相比：痛风康治疗组第4天、第5天患者自评关节疼痛评分明显下降(第4天：P<0.05；第5天：P<0.01),第3天、第6天患者关节压痛减轻(均P<0.05),第3天、第6天关节急性红肿减轻(均P<0.05),第6天关节活动受限程度减轻(P<0.05),使用止痛药的情况较对照组减少(P<0.05)
(2)痛风康治疗组患者谷丙转氨酶、肌酐、尿素氮等安全性指标治疗前后均无明显变化(谷丙转氨酶：P>0.05；肌酐：P>0.05；尿素氮P>0.05)
(3)治疗后痛风康治疗组血清TREM-1浓度明显低于对照组(P<0.05)。痛风康治疗组血清TREM-1浓度下降幅度较对照组显著,对照组下降了28.9%,而治疗组下降了42.4%。
实验研究：
(1)与正常组相比,模型组造模后4、10、24、72小时关节肿胀均显著增加(4h：P<0.01；10h：P<0.01；24h：P<0.01；72h：P<0.01)。与模型组相比,秋水仙碱组造模后4、10、24、72小时关节肿胀均显著减轻(4h：P<0.05；10h：P<0.05；24h：P<0.05；72h：P<0.01)；痛风康高、中、低剂量组造模后4、10、24小时关节肿胀有减轻的趋势,72小时关节肿胀均显著减轻(痛风康高剂量组：P<0.01；痛风康中剂量组：P<0.01；痛风康低剂量组：P<0.01),与秋水仙碱组比较无统计学差异(痛风康高剂量组：P>0.05；痛风康中剂量组：P>0.05；痛风康低剂量组：P>0.05),痛风康高、中剂量组关节肿胀指数有较低剂量组降低的趋势。
模型组10h表现出明显的关节炎症红肿,到24h、72h表现出逐步缓解的趋势；与模型组比较,秋水仙碱组、痛风康低、中、高剂量组关节炎症红肿明显减轻,到72h关节仅表现轻度肿胀。
模型组10h表现出明显的关节功能障碍,到24h、72h逐步缓解；与模型组比较,秋水仙碱组、痛风康低、中、高剂量组关节功能障碍明显减轻,到72h关节功能障碍基本消失。
(2)HE染色显示模型大鼠关节滑膜炎症明显,滑膜细胞增生,伴炎细胞浸润,小血管增生及成纤维细胞,提示造模成功。经痛风康和秋水仙碱治疗后,上述病理改变减轻。
(3)正常组大鼠关节滑膜组织可见极少的TLR2表达。与正常组相比,模型组大鼠关节滑膜组织TLR2表达明显增多(P<0.01)。与模型组相比,秋水仙碱组滑膜组织TLR2表达明显减少(P<0.01),痛风康低、中、高剂量组滑膜组织TLR2表达明显减少(痛风康低剂量组：P<0.0)；痛风康中剂量组：P<0.01；痛风康高剂量组：P<0.01)痛风康低、中、高剂量组TLR2表达呈递减趋势。与秋水仙碱组比较,痛风康低、中、高剂量组滑膜组织TLR2表达无明显差别。
(4)正常组大鼠关节滑膜组织可见极少的TLR4表达。与正常组相比,模型组大鼠关节滑膜组织TLR4表达明显增多(P<0.01)。与模型组相比,秋水仙碱组滑膜组织TLR4表达明显减少(P<0.05),痛风康低、中、高剂量组滑膜组织TLR4表达明显减少(痛风康低剂量组：P<0.01,痛风康中剂量组：P<0.05,痛风康高剂量组：P<0.01)。与秋水仙碱组比,痛风康低、中、高剂量组滑膜组织TLR4表达无统计学差异。
(5)正常组大鼠关节滑膜组织可见较少的MyD88表达。与正常组相比,模型组大鼠关节滑膜组织MyD88表达明显增多(P<0.01)。与模型组相比,秋水仙碱组滑膜组织MyD88表达明显减少(P<0.01),痛风康低、中、高剂量组滑膜组织MyD88表达均减少(痛风康低剂量组：P<0.01,痛风康中剂量组：P<0.01,痛风康高剂量组：P<0.01),痛风康高、中剂量MyD88表达有较低剂量组进一步降低的趋势。与秋水仙碱组比,痛风康低、中、高剂量组滑膜组织MyD88表达无统计学差异。
结论
(1)痛风康可明显改善急性痛风性关节炎患者关节疼痛、红肿及功能障碍,且用药安全,对患者肝肾功能无明显影响。
(2)痛风康可明显改善MSU致急性痛风性关节炎模型大鼠的关节肿胀、红肿及功能障碍,改善滑膜组织炎症病理变化。
(3)痛风康可明显下调急性痛风性关节炎患者血清TREM-1的表达,对TREM-1表达的抑制可能是痛风康治疗急性痛风性关节炎的作用机制之一。
(4)痛风康可明显下调MSU致炎症关节滑膜组织的TLR2、TLR4、MyD88的表达,对TLRs/MyD88通路的干预,可能是痛风康治疗急性痛风性关节炎的作用机制之一。
Background
With the social and economic development, people's diet and lifestyle change greatly. Furthermore, aging of the population in the world has become more evident especially in China, so the prevalence of gout shows an increasing trend generally. It poses a threat to people's health. Because acute gouty arthritis leads to the repeated attack of pain, it will easily disabled, teratogenic, and affect joint function if not treated actively. Patients with acute gouty arthritis occupied the biggest percentage in visiting patients with gout. Prevention and treatment of acute gouty arthritis should be studied and solved urgently. Traditional Chinese Medicine(TCM) has been long for prevention and treatment of gout. TCM is rich in theory and effective herbs in the treatment of gout, so it is of great significance and prospects for further research and development of effective prescriptions.
According to therapeutic thoughts of reinforcing Kidney and activating blood, eliminating heat and wetness evil, Professor Liu Youzhang created the TCM compound formula named as TongFengKang(TFK) on the basis of long-term clinical practice. Previous clinical researchs have confirmed preliminary that effect of TFK on the acute gouty arthritis. TFK not only can improve patients'joint symptoms with acute gouty arthritis, but also can effectually decrease uric acid in patients'blood. Preliminary animal experiments have shown that TFK can reduce inflammatory cells infiltration, relieve the damage of synovial tissue, and reduce hyperemia in model rats with acute gouty arthritis. TFK have significant effect on swelling inhibition and analgesia, but its action mechanism needs to be further clarified.
Recent investigations provided novel evidence in the pathology of acute gout. A number of studies indicated that MSU crystals could act as a "danger signal" which resembled exogenous adjuvants, and toll-like receptors(TLRs) could recognize such "danger signals". Myeloid differentiation factor 88(MyD88) was a key adapter molecule in TLRs signaling pathway, which had an important role in passing the upstream signal and the development of disease. MyD88-dependent pathway was the common pathway of TLRs signals. In addition, TLRs were involved in triggering receptors expressed on myeloid cells 1 (TREM-1) pathways. Activation of TLRs could lead to up-regulation of TREM-1, and TREM-1 expression could change the expression of the key receptor and effector proteins in TLR4 pathway. Thus, TREM-1 and TLRs pathways might have a synergistic effect. Observing the effect of TFK on TLR2, TLR4, MyD88, TREM-1 will be beneficial to further reveal it action mechanism.
Objectives
Study therapeutic effect and security of TFK on treatment of acute gouty arthritis, and observe the effect of TFK on expression of patients'serum (TREM-1) with acute gouty arthritis; Research the effect of TFK on joint symptom and TRL2, TLR4, MyD88 in rats synovial tissues with acute gouty arthritis, then further confirm efficacy of TFK and reveal its action mechanism.
Methods
Clinical research:
40 patients with acute gouty arthritis were randomly divided into two groups:treatment group and control group,20 patients in each group. The treatment group was treated by "TFK granule" and "Tongfeng external application", the control group was treated with placebo. The treatment duration was 5 days. Recorded the index of the patients'joint pain, acute swelling and dysfunction. Recorded self-rating joint pain index. Detected patients'hepatic and renal function in pre and post treatment. Testing the expression of TREM-1 in patients'serum before and after the treatment by enzyme linked immunosorbent assay(ELISA). Experimental study:
48 rats were randomly divided into normal group, model group, colchicine group, large dose TFK group, medium dose TFK group, small dose TFK group,8 rats in each group. TFK large, medium and small dose group were given TFK separately in the dose of 72g/kg per day,36g/kg per day,18g/kg per day. Colchicine group was given colchicine solution in the dose of 0.3mg/kg per day. Saline water was administered to the normal group and model group. All rats had been oral administered by the drugs for 5 days. On the third day of drug, acute gouty arthritis model was set up via articular cavity MSU injection except the normal group, the same volume of saline was injected to the normal group. Right ankle joint circumference of rats was measured dynamically. The indexes of rats'inflammation and dysfunction were observved. The pathological changes of rats joint synovial tissue were detected by HE staining. The expression of TLR2, TLR4, MyD88 in rats synovium were measured by immunohistochemistry.
Results
Clinical research:
(1)Compared with control group, self-rating joint pain index of TFK treated group was significantly lightened(day4:P<0.05; day5:P<0.01), articular pain was lightened (day 3:P<0.05; day 6:P<0.05), acute swelling was lightened (day 3:P<0.05; day 6:P<0.05), dysfunction was lightened(day6:P<0.05), analgesic use decreased(P<0.05).
(2)Security indexes such as alanine aminotransferase(ALT), urea nitrogen (BUN), creatinine(CREA) of TFK treated group did not show significant change in pre and post treatment(ALT:P>0.05;BUN:P>0.05;CREA:P>0.05).
(3) Compared with control group, TREM-1 concentration of TFK treated group decreased significantly after treatment(P<0.05).The decrease before and after treatment of TFK treated group was more significant(decreased by 42.4%), whereas 28.9% in control group. Experimental study:
(1)Compared with normal group, the joint swelling index of model group was significantly increased(4h after model:P<0.01; 10h:P<0.01;24h:P<0.01; 72h: P<0.01). Compared with model group, the joint swelling index of colchicines group was significantly lightened(4h:P<0.05; 10h:P<0.05;24h:P<0.05; 72h: P<0.01); The joint swelling index of TFK large, medium and small dose group showed a decreasing trend at 4h, 10h and 24h, but was significantly lightened (TFK large dose group:P<0.01, TFK medium dose group:P<0.01 and TFK small dose group:P<0.01), but did not show significant change compared with colchicines group9 (TFK large dose group:P>0.05, TFK medium dose group:P>0.05 and TFK small dose group:P>0.05), and the joint swelling index of TFK large and medium dose group showed a decreasing trend compared with small dose group.
Model group showed significant joint inflammation(10h), and a gradually catabatic trend(24h,72h); Compared with model group, the joint inflammation of colchicines group, TFK small, medium and large dose group was significantly lightened, and was slight swelling at 72h.
Model group showed significant joint dysfunction(10h), and a catabatic gradually trend(24h,72h); Compared with model group, the joint dysfunction of colchicines group, TFK small, medium and large dose group was significantly lightened, and almost disappeared at 72h.
(2)The abnormalities of the rat's joint synovial tissue included obvious inflammation, synovial cells proliferation, inflammatory cell infiltration, small vessels proliferation, fibroblast cell by HE staining, which shown the animal model was successfully produced. After the treatment TFK and colchicines, these histological abnormalities were lessened.
(3)The TLR2 very few expression in joint synovial tissue of normal group. Compared with normal group, The TLR2 expression in joint synovial tissue of model group increased significantly(P<0.01). Compared with model group, The TLR2 expression in joint synovial tissue of colchicines group decreased significantly (P<0.01), The TLR2 expression in joint synovial tissue of TFK small, medium and large dose group decreased significantly(TFK small dose group:P<0.01; TFK medium dose group:P<0.01; TFK large dose group:P<0.01). TFK small, medium and large dose group showed a decreasing trend. Compared with colchicines group, The TLR2 expression in joint synovial tissue of TFK small, medium and large dose group did not show significant change.
(4)The TLR4 very few expression in joint synovial tissue of normal group. Compared with normal group, The TLR4 expression in joint synovial tissue of model group increased significantly(P<0.01). Compared with model group, The TLR4 expression in joint synovial tissue of colchicines group decreased significantly (P<0.05), The TLR2 expression in joint synovial tissue of TFK small, medium and large dose group decreased significantly(TFK small dose group:P<0.01; TFK medium dose group:P<0.05; TFK large dose group:P<0.01). Compared with colchicines group, The TLR4 expression in joint synovial tissue of TFK small, medium and large dose group did not show significant change.
(5)The MyD88 less expression in joint synovial tissue of normal group. Compared with normal group, The MyD88 expression in joint synovial tissue of model group increased significantly(P<0.01). Compared with model group, The MyD88 expression in joint synovial tissue of colchicines group decreased significantly(P<0.01), The MyD88 expression in joint synovial tissue of TFK small, medium and large dose group decreased significantly(TFK small dose group:P<0.01;TFK medium dose group:P<0.01; TFK large dose group: P<0.01). The MyD88 expression of TFK large and medium dose group showed a decreasing trend compared with small dose group. Compared with colchicines group, The TLR4 expression in joint synovial tissue of TFK small, medium and large dose group did not show significant change.
Conelusion
(1) TFK not only can significantly improve joint pain, swelling, dysfunctiong of acute gouty arthritis, but also was safe, there was no obvious impact on hepatic and renal function.
(2)TFK can significantly improve joint pain, swelling, dysfunctiong of rat with MSU-induced acute gouty arthritis.
(3)TFK can significantly reduce the expression of TREM-1 in patients'serum with acute gouty arthritis. The inhibition effect of TFK on the expression of TREM-1 was one of TFK mechanism in acute gouty arthritis treatment.
(4)TFK can significantly reduce the TLR2, TLR4 and MyD88 expression of synovium from inflammatory arthritis. The regulatory effect of TFK on TLRs/MyD88 pathways was one of TFK mechanism in acute gouty arthritis treatment.
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