1.成都中医药大学针灸推拿学院;2.成都中医药大学养生康复学院
四川省自然科学基金面上项目（NO.2024NSFSC0729）；国家自然科学基金（NO.82105027、82205239）
1.School of Acu-Mox and Tuina,Chengdu University of Traditional Chinese Medicine;2.School of Health Rehabilitation,Chengdu University of Traditional Chinese Medicine
目的：基于微小RNA(micro-RNA,miRNA)高通量测序技术,探究艾灸改善骨质疏松模型大鼠的分子机制。方法：18只雌性SD大鼠随机分为假手术组(6只)和手术组(12只)。手术组以去卵巢手术法制备骨质疏松症(osteoporosis,OP)模型,将造模成功的12只大鼠随机分为模型组和艾灸组,每组6只。艾灸组大鼠艾灸双侧“肾俞”、“关元”穴,每日1次,每日20min,持续12周。Micro-CT扫描大鼠股骨并成像,分析骨小梁厚度(trabecular thickness,Tb.Th)、骨小梁分离度(trabecular separation,Tb.Sp)、骨体积分数(bone volume/total volume,BV/TV)；HE染色观察大鼠胫骨组织形态；ELISA法检测血清碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,OCN)含量；随机挑选3只大鼠的右胫骨进行miRNA高通量测序,筛选差异表达的miRNA,并对差异表达miRNA进行功能富集分析与靶基因预测。结果：Micro-CT图像显示艾灸组骨密度优于模型组,与模型组比较,艾灸组Tb.Th厚度显著增加,BV/TV分数显著上升,Tb.Sp显著下降(P＜0.05)；ELISA结果表明,与模型组比较,艾灸组血清ALP活性显著降低,血清OCN含量显著上升(P＜0.05)；miRNA测序结果显示模型组上调基因与干预后下调基因共同表达34个,模型组下调与干预后上调基因共同表达15个。KEGG通路富集分析结果显示,差异基因的表达主要富集于MAPK、Ras、FoxO、TNF、癌症中的miRNA等信号通路途径。对显著性最高的前5个差异miRNA,miR-153-5p、miR-201-5p、miR-449c-5p、miR-451-3p、miR-153-3p的靶基因预测得到10个主要靶点,Ebf2、Rtn4、Fbxl3、Naa15、Vamp2、Daam1、Akap6、Camta1、Ptprz1、Lamp1。结论：艾灸能够延缓OP病程、改善骨微结构、平衡骨代谢,这种治疗作用可能通过调控miRNA及其靶基因表达来实现,更详细的机制有待进一步探索。
Objective: Based on high-throughput sequencing technology for micro-RNA (miRNA), this study explores the molecular mechanisms of moxibustion in improving osteoporosis in rats. Methods: A total of 18 female SD rats were randomly divided into a sham operation group (6 rats) and an operation group (12 rats). The operation group was subjected to ovariectomy to induce osteoporosis (OP), and the 12 rats with successful modeling were randomly divided into a model group and an moxibustion group, with 6 rats in each group. The moxibustion group rats were treated with moxibustion at the “Shenshu” (BL23) and “Guanyuan” (CV4), once a day, for 20 minutes each time, for 12 weeks. Micro-CT scan the rat femur and image it, analyze the trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and bone volume fraction (BV/TV) of the trabecular bone, and HE staining was used to observe the morphology of tibia tissue. Serum alkaline phosphatase (ALP) and osteocalcin (OCN) levels were measured using ELISA. Three randomly selected rats from each group were used for miRNA high-throughput sequencing, and differentially expressed miRNAs were screened. The functional enrichment analysis and target gene prediction were performed for the differentially expressed miRNAs. Results: The Micro-CT images showed that the bone density of the moxibustion group was superior to that of the model group, and compared to the model group, the moxibustion group showed a significant increase in the thickness of the trabecular bone (Tb.Th), a significant rise in the bone volume/tissue volume (BV/TV) score, and a significant decrease in the trabecular separation (Tb.Sp) (P < 0.05). The ELISA results indicated that compared to the model group, the moxibustion group showed a significant decrease in serum ALP activity and a significant increase in serum OCN content (P < 0.05). The miRNA sequencing results showed that 34 miRNAs were commonly expressed between the model group and the intervention group, and 15 miRNAs were commonly expressed between the model group and the intervention group. The KEGG pathway enrichment analysis showed that the expression of different genes was mainly enriched in signaling pathways such as MAPK, Ras, FoxO, TNF, and cancer-related microRNAs. For the top 5 differentially expressed microRNAs with the highest significance, namely miR-153-5p, miR-201-5p, miR-449c-5p, miR-451-3p, and miR-153-3p, target gene predictions yielded 10 major targets: Ebf2, Rtn4, Fbxl3, Naa15, Vamp2, Daam1, Akap6, Camta1, Ptprz1, and Lamp1. Conclusion: Acupuncture can slow down the progression of OP, improve bone microstructure, and balance bone metabolism. This therapeutic effect may be achieved by regulating the expression of microRNAs and their target genes, and a more detailed mechanism awaits further exploration.
