课题组已确定HMGB1/LPS-NF-κB-Klotho轴具有调控大肠癌生物学的功能。在此基础上，预实验发现大肠癌组织和细胞系中TLR-4和Klotho的表达呈负相关，TLR-4是HMGB1和LPS的共同受体。因此，HMGB1和LPS可能通过HMGB1/LPS-TLR-4-NF-κB-Klotho轴调控Klotho基因的表达。用含有天马颗粒Ⅲ的大鼠中药血清干预大肠癌细胞系，细胞凋亡增加，TLR-4表达下调，Klotho表达上调。我们推测天马颗粒Ⅲ可能通过TLR-4/Klotho途径调控大肠癌的生物学功能。本课题运用流式细胞技术、RT-PCR、免疫印记等方法研究天马颗粒Ⅲ抗肿瘤的分子机制，完善了复方通过多角度多层次抗肿瘤的理论依据，并为该信号通路提供有效的抑制药物，是对既往前期研究的延续和深化。

Our previous findings that HMGB1/LPS-NF-κB-Klotho pathway regulated the phenotypes of colorectal cancer. Our preliminary study found that TLR-4, a common receptor for HMGB1 and LPS, was highly expressed, whereas the expression of klotho gene was lowered in colorectal cancer tissues and cell lines. We therefore hypothesized that HMGB1 and LPS may regulate the expression of klotho gene through the HMGB1/LPS-TLR-4-NF-κB-Klotho pathway. In addition, the serum extracted from rats feed with Tianma Granules Ⅲ significantly induced apoptosis, down-regulated the expression of TLR-4, but up-regulated the expression of klotho gene in colorectal cancer cells. We therefore hypothesized Tianma Granules Ⅲ may regulate the phenotypes of colorectal cancer cells through TLR-4/Klotho pathway. Using flow cytometry, RT-PCR and immunoblotting approaches, we will investigate the anti-tumor molecular mechanisms of Tianma Granules Ⅲ. The proposed studies will provide the theoretical basis for the anti-tumor effect of Tianma compound at multi-angle and multi-levels, and the development of agents to interfere the signal pathway. This proposed study is an extended and deepen exploration of our previous findings.