基于TLR2信号通路研究温针灸治疗膝骨关节炎肾阳虚证的免疫效应机制

In syndrome-genome platform supported by National Natural Science Fund, it was found that the differentially expressed genes of kidney-yang deficiency syndrome were related with immune function, especially the genes in TLR2(Toll-like receptor 2 )signal pathway which was inferred having close relationship with kidney-yang deficiency syndrome. The TLR2 signal pathway was chosen in this topic to research the kidney-yang deficiency syndrome’s complex pathological basis. 20 patients suffering from osteoarthritis of the knee with kidney-yang deficiency syndrome were extracted total RNA from peripheral blood . The gene expression profile were compared between experimental groups using cDNA microarray.The acupoints of Shenshu,Guanyuan,Zusanli, Ashixue and Yanglingquan were elected for warm-needle acupuncture for the patients. Patients with remarkable effect were selected to compare the gene expression profile before and after warm-needle acupuncture using cDNA microarray. The key genes’ expression level in TLR2 signal pathway, such as TLR2, MyD88, and IL-1β was verified using q-PCR technique to screen the marker genes. Western blotting technique was used to detect the key enzyme content in the pathway and measure the enzyme activity. The relationship between TLR2 pathway and kidney-yang deficiency syndrome and the regulation mechanism of warm-needle acupuncture were explored in this research through information fusion and data processing. This study will provide theoretical and experimental basis for kidney-yang deficiency syndrome’s in-depth research, and provide clinical diagnosis and treatment of osteoarthritis of the knee, and provide model example for combination of disease and syndrome’s transcriptome study.

在课题组国家自然科学基金证候-基因组平台上发现肾阳虚证差异表达基因集中于免疫类，尤其是TLR2（Toll-like receptor）通路基因，提示它与肾阳虚证发生关系密切。本课题切入TLR2通路，筛选典型膝骨关节炎肾阳虚证患者20例，提取血液总RNA进行基因芯片检测，并与肾阴虚骨关节炎患者、正常人比较差异基因表达谱；取肾腧、关元、足三里、阿是穴和阳陵泉等穴位对膝骨关节炎肾阳虚证患者进行温针灸治疗，对显效患者进行治疗后芯片检测。比较各实验组芯片结果，对TLR2通路TLR2、MyD88、IL-1β等关键基因进行q-PCR验证，用Western blotting等技术检测通路关键酶含量，并检测其活性；通过信息融合数据处理，探讨TLR2通路在肾阳虚证发生及温针灸治疗中的调控机制、关键作用环节。本研究将为肾阳虚证病理基础深入研究、膝骨关节炎临床诊治提供理论和实验依据，为病证结合的转录组学提供典范。

肾阳虚证是中医脏腑辨证的一个重要基础证型，见于多种疾病的过程中，其症状表现相对稳定和持久，因此肾阳虚证的现代研究备受关注。膝骨关节炎（knee osteoarthritis ,KOA）是中老年人的常见病、多发病，针灸治疗KOA是中医骨伤的特色疗法之一，具有简便、经济、疗效确切等优势。本研究采用病证结合的方法，通过对KOA进行证候调查，筛选典型KOA肾阳虚证病例30例，选择肾俞、关元、足三里为主穴，阳陵泉及阿是穴为配穴进行2个疗程的温针灸治疗。对疗效显著的10例病例进行治疗前后的基因芯片检测，并与KOA肾阴虚证组及健康对照组进行对比，对筛选到的差异表达基因进行GO、KEGG通路富集分析及qPCR验证。研究结果如下：（1）证候调查显示KOA的常见证型为肝肾亏虚证、痰瘀阻络证、痰湿闭阻证、肾阳虚证等，肾阳虚证所占比例为23.08%。（2）筛选病例温针灸治疗的KOA临床有效率为93.33%，中医证候疗效有效率达96.67%，治疗后膝关节疼痛、僵硬、功能以及肾阳虚症状得到明显改善。（3）KOA肾阳虚证治疗前组VS健康对照组差异表达基因有98条，主要富集于免疫相关的GO类及信号通路；筛选到KOA肾阳虚证治疗后VS健康对照组差异表达基因31条；温针灸治疗后组VS温针灸治疗前组共筛选到差异表达基因52条，主要富集于代谢相关GO及KEGG通路，治疗前与健康对照组富集的免疫功能相关通路未被筛选到。（4）qPCR验证实验：KOA肾阳虚证治疗前组VS健康对照组筛选到的TNF、CCL3、KLRC3和JUN基因，组间表达趋势跟芯片结果一致。KOA肾阳虚证治疗后VS治疗前筛选到的ACSM5、ALAS2、HBM、GABRP、HBA2、STRADB和SYNE1的表达趋势与芯片结果一致，ZNF83验证结果与芯片相反。qPCR验证结果与芯片结果吻合度较高。本研究结果提示KOA肾阳虚证的关键病机之一是机体免疫功能的紊乱；温针灸治疗后病例部分异常差异表达基因得到纠正或改善，治疗前后差异表达基因主要富集于代谢类，提示温针灸的疗效机制可能是通过调节机体代谢来改善机体免疫功能。