温针灸干预膝骨关节炎骨重塑及其对Ddit4/mTOR自噬通路的调控机制

The process of bone remodeling is the key to maintain the integrity of the skeletal structure. And there are abnormal bone remodeling in the early stage of knee osteoarthritis (KOA), bone loss is the main aspects. It’s helpful to prevent or delay the occurrence and development of KOA by maintaining the homeostasis of bone remodeling. The activation of mTOR- autophagy pathway was believed playing key role in bone remodeling. Warm-needle acupuncture has a good therapeutic effect on KOA. The preliminary work has shown that the preliminary effect of Warm-needle acupuncture intervene on bone remodeling in KOA, and we use the Gene expression profiles to screen out the KOA differential gene Ddit4 by the treatment of Warm-needle acupuncture. It is speculated that Warm-needle acupuncture may play its role via Ddit4/mTOR autophagy pathway. On the basis of the previous work, this project intends to establish the model of KOA to observe the bone remodeling and the relativity between Ddit4/mTOR autophagy pathway and bone remodeling by using micro-CT, and it also validated them by pharmacological methods. Through the intervention at different time points, we observe the intervention effect on bone remodeling and its role in the regulation of Ddit4/m-TOR antophagy pathway so as to clarify the advantages of Warm-needle acupuncture early intervention of KOA’s bone remodeling, and ulteriorly verify the mechanism of Warm-needle acupuncture in regulating bone remodeling via Ddit4/mTOR autophagy pathway by pharmacological methods. The purpose of this study was to explore the effect and mechanism of Warm-needle acupuncture intervening on bone remodeling in KOA , and provide a scientific basis and explanation for the treatment and the occasion of Warm-needle acupuncture intervention of KOA, in order to promote the wider clinical application of acupuncture and moxibustion in the prevention and treatment of KOA.

骨重塑过程是维持骨骼结构完整的关键，KOA早期即存在骨重塑异常，以骨丢失为主。mTOR-自噬通路被认为是骨重塑的关键环节。前期工作显示温针灸干预KOA骨重塑的初步效应，利用基因芯片筛选出温针灸治疗KOA差异基因Ddit4，推测温针灸可能通过Ddit4/mTOR自噬通路发挥该效应。本项目拟建立KOA模型，观察骨重塑情况，探索Ddit4/mTOR自噬通路与骨重塑的相关性，并使用药理学手段进行验证。通过不同时间点温针灸干预介入，观察温针灸对骨重塑的干预效应及其对Ddit4/mTOR自噬通路的调控作用，明确温针灸早期干预KOA骨重塑的优势；进一步用药理学手段验证温针灸通过Ddit4/mTOR自噬通路调控骨重塑的机制。通过探索温针灸对骨重塑的调控作用及机制，尝试从新的角度探讨温针灸对KOA的防治效应和机制，为温针灸防治KOA及其治疗时机选择提供科学依据和阐释，为中医“治未病”提供部分实验依据。

骨重塑过程是维持骨骼结构完整的关键，KOA发生初期即存在骨重塑异常，以软骨下骨高骨转换为背景的骨重塑是OA骨赘形成过程中的启动因素，早期干预软骨下骨骨重塑可能是防止或延缓OA发生、发展的潜在有效途径。温针灸是临床治疗KOA广泛采用的治疗手段，初步发现温针灸早期干预能够减轻KOA的病理损伤，但有待进一步明确其早期干预的效应优势，研究表明mTOR与自噬均参与骨重塑过程，并受Ddit4调制。本项目拟通过不同时间点干预，进一步明确超早期温针灸治疗对软骨下骨骨重塑的效应优势；在此基础上，检测Ddit4、mTOR及自噬蛋白LC3-Ⅱ、Beclin1水平，探索温针灸基于Ddit4/mTOR自噬通路对骨重塑的调控机制。主要研究结果：（1）KOA初期（1周）软骨下骨呈现高骨转换，骨量丢失，继之出现骨形成增加，软骨下骨的骨密度增加；温针灸超早期（在造模后立即进行干预）较造模1周后干预可以促进骨密度增加，骨小梁排列变整齐有序，骨体积分数、骨小梁密度、骨矿物质含量、骨小梁数量升高（P<0.001），骨小梁分离度降低（P<0.001），提示温针灸超早期干预对骨重塑的效应优势；（2）与模型组比较，Ddit4抑制剂组能增加软骨下骨的骨密度，其中骨体积分数、骨小梁密度、骨矿物质含量、骨小梁厚度均升高（P < 0.001）；（3）KOA建模1周后出现骨钙素降低，PINP升高，βCTX升高（P < 0.001），4周及8周时出现骨钙素、MMP-3升高，PINP、βCTX降低（P < 0.001）；温针灸超早期治疗改善骨代谢指标，促进骨钙素增加，降低PINP、βCTX；（4）PCR结果显示：与空白组比较，KOA建模1周、4周和8周Ddit4、LC3I和LC3II表达量降低，mTOR和Beclin 1增加。温针灸超早期治疗后Ddit4和LC3增加，mTOR和Beclin 1减少，与空白组比较未见明显差异（P>0.05）。WB结果显示温针灸超早期治疗可升高Ddit4的表达，降低mTOR。通过本项目研究阐明超早期温针灸干预可以改善KOA软骨下骨骨重塑，可能与Ddit4/mTOR自噬信号通路的调控有关，为温针灸干预KOA及治疗时机选择提供科学依据，体现中医“治未病”的优势，以期推动温针灸更广泛地防治KOA。