姜黄素通过抑制RANBP3L基因表达减轻2型糖尿病小鼠的骨质疏松症

doi:10.12122/j.issn.1673-4254.2026.02.10

摘要/Abstract

摘要：

目的探讨姜黄素通过影响Ran结合蛋白3样（RANBP3L）的表达对2型糖尿病（T2DM）引起的骨质疏松的治疗作用。方法取MC3T3-E1细胞，成骨诱导培养后随机分为以下4组：对照组（Control），高糖处理组（HG），姜黄素组（Cur），高糖处理+姜黄素组（HG+Cur），药物预先干预24 h。CCK-8法筛选姜黄素最佳干预浓度；碱性磷酸酶（ALP）和茜素红S（ARS）染色检测成骨分化和矿化相关标志物；采用RNA-seq测序分析姜黄素干预后高糖下培养MC3T3-E1成骨细胞转录特征。体内实验中，通过高脂饮食（HFD）和链脲霉素（STZ）注射建立T2DM小鼠模型，20只8周龄雄性C57BL/6小鼠随机分为4组：正常+溶剂组（Ctrl+Veh）、T2DM模型+溶剂组（T2DM+Veh）、T2DM模型+ RANBP3L敲低+溶剂组（T2DM+KD+Veh）、T2DM模型+ RANBP3L敲低+姜黄素组（T2DM+KD+Cur），5只/组。T2DM+KD+Cur组每日给予58 mg/kg的姜黄素灌胃处理，其余各组给予等量溶剂灌胃。ELISA检测小鼠血脂水平；HE染色观察骨微结构特征；茜素红S染色检测钙盐沉积情况；免疫组化染色检测ALP、RANBP3L表达；Q-PCR和Western blotting检测小鼠骨组织中RANBP3L、OCN、RANKL表达，及TNF-α/NF-κB通路相关蛋白表达情况。结果姜黄素可促进高糖环境下MC3T3-E1细胞的增殖和成骨分化，下调RANBP3L蛋白的表达（P<0.01）。体内研究表明，当RANBP3L基因敲减后，模型小鼠的骨微结构增强，成骨标志物水平提高，血糖浓度降低，血脂平衡改善，且TNF-α/NF-κB信号通路相关蛋白受抑制（P<0.05），联用姜黄素后可进一步降低小鼠骨组织中RANBP3L的表达并加强治疗效果（P<0.05）。结论姜黄素可通过降低RANBP3L的表达，促进成骨分化能力，缓解糖脂代谢紊乱，减轻二型糖尿病骨质疏松症，可能通过抑制TNF-α/NF-κB信号通路发挥作用。

关键词: 姜黄素, RANBP3L, 2型糖尿病骨质疏松, RNA转录组测序

Abstract:

Objective To investigate the therapeutic mechanism of curcumin for osteoporosis induced by type 2 diabetes mellitus (T2DM). Methods In cultured MC3T3-E1 osteoblasts exposed to high glucose (HG), the effects of curcumin treatment on osteogenic differentiation and mineralization were examined by assessing alkaline phosphatase (ALP) activity and using alizarin red S (ARS) staining. RNA-seq sequencing was used to analyze the transcriptional characteristics of MC3T3-E1 osteoblasts in HG culture after curcumin treatment. In a mouse model of T2DM established by high-fat diet feeding and streptococcal injection, the effects of solvent vehicle, vehicle+RANBP3L knockdown, and RANBP3L knockdown+curcumin treatment on blood lipids, bone microstructure and calcium deposition was evaluated using ELISA, HE staining, and Alizarin red S staining. Osteogenic differentiation ability of mouse femoral tissues were assessed by detecting ALP, RANBP3L, OCN and RANKL expressions using immunohistochemical staining, RT-qPCR and Western blotting, and the changes in the TNF‑α/ NF‑κB pathway were detected with Western blotting. Results Curcumin obviously promoted proliferation and osteogenic differentiation of MC3T3-E1 cells in HG culture and down-regulated cellular expression of RANBP3L protein. In the diabetic mouse models, RANBP3L knockdown significantly improved bone microstructure and blood lipid balance, increased expression levels of osteogenic markers, decreased blood glucose level, and caused inhibition of the TNF‑α/NF‑κB signaling pathway. The combined treatment with curcumin further reduced RANBP3L expression levels in the bone tissue and significantly enhanced the therapeutic effect. Conclusion Curcumin promotes osteogenesis, alleviates glucose and lipid metabolism disorders, and improves osteoporosis in type 2 diabetic mice possibly by inhibiting TNF‑α/NF‑κB signaling pathway via suppressing RANBP3L expression.

Key words: curcumin, RANBP3L, type 2 diabetes osteoporosis, mRNA sequencing

邹晓松, 张幸, 李平, 田瑞雪, 路晓淼. 姜黄素通过抑制RANBP3L基因表达减轻2型糖尿病小鼠的骨质疏松症[J]. 南方医科大学学报, 2026, 46(2): 325-334.

Xiaosong ZOU, Xing ZHANG, Ping LI, Ruixue TIAN, Xiaomiao LU. Curcumin improves osteoporosis in type 2 diabetic mice by inhibiting RANBP3L expression[J]. Journal of Southern Medical University, 2026, 46(2): 325-334.

图/表 7

表1 实时聚合酶链反应引物序列

Tab.1 Primer sequences for RT-qPCR

Gene	Primer sequences (5'to3')	Size (bp)
GAPDH-m	F:AGGTCGGTGTGAACGGATTTG	95
GAPDH-m	R:GGGGTCGTTGATGGCAACA	95
RANBP3L	F:GAGAAAGCGTGTAAGGTCTTCA	82
RANBP3L	R:CGTTTGAGACAAAGTTGCCAC	82
OCN	F:AGTGTGAGCTTAACCCTGCT	156
OCN	R:GAGGATCAAGTCCCGGAGAG	156
RANKL	F:CGCTCTGTTCCTGTACTTTCG	114
RANKL	R:GAGTCCTGCAAATCTGCGTT	114

图1 姜黄素促进高糖条件下MC3T3-E1细胞的增殖及成骨分化

Fig.1 Curcumin promotes proliferation and osteogenic differentiation of MC3T3-E1 cells in high-glucose (HG) culture. A： CCK-8 assay for assessing cell viability following HG culture and treatment with different concentrations of curcumin. B： ALP staining of the cells after curcumin treatment for 7 days (Scale bar=50 μm). C： Alizarin red staining of the cells after curcumin treatment for 14 days. D： Relative area of ALP staining (Scale bar=50 μm). E： Relative area of Alizarin red staining. n=3. *P<0.05 vs HG group, **P<0.01, ***P<0.001 vs Control; ##P<0.01, ###P<0.001 vs HG.

图2 姜黄素可降低高糖条件下MC3T3-E1细胞RANBP3L的表达

Fig.2 Curcumin reduces RANBP3L expression in MC3T3-E1 cells cultured in high glucose. A: Volcano plot showing the distribution of differentially expressed mRNAs. B: KEGG pathway analysis of the pivotal signaling pathways within the differentially expressed genes. C: mRNA expression of RANBP3L detected by qRT-PCR. D, E: Protein expression of RANBP3L detected by Western blotting. n=3. *P<0.05, ***P<0.001 vs Control; ##P<0.01, ###P<0.001 vs HG.

图3 各组小鼠的血糖和血脂水平

Fig.3 Blood glucose and lipid profiles of the mice in each group. A: Schematic diagram of the experimental process. B: Fasting blood glucose of the mice after STZ injection. C-F: TC, TG, LDL-C and HDL-C at the 17rd week. n=5. **P<0.01, ***P<0.001 vs Ctrl+Veh; ##P<0.01, ###P<0.001 vs T2DM+Veh; ‡P<0.05, ‡‡P<0.01 vs T2DM+KD+Veh.

图4 糖尿病模型小鼠表现出骨形成减少

Fig.4 Diabetic mice show reduced bone formation. A： HE staining of the bone tissues (Scale bar=200 μm). B： Alizarin Red Staining of the bone tissues (Scale bar=200 μm). C, D： Immunohistochemistry for ALP and RANBP3L （Scale bar=100 μm）. E-G： mRNA expression of RANBP3L, OCN and RANKL detected by qRT-PCR. H-J： Protein expressions of OCN and RANKL detected by Western blotting. n=5. ***P<0.001 vs Ctrl+Veh.

图5 姜黄素下调RANBP3L促进T2DM小鼠骨形成

Fig.5 Curcumin down-regulates RANBP3L expression and promotes bone formation in T2DM mice. A： HE staining of the bone tissues (Scale bar=200 μm). B： Alizarin Red Staining of the bone tissues (Scale bar=200 μm). C-F： Immunohistochemistry for ALP and RANBP3L and quantitative histograms (Scale bar =100 μm). G, I and J： mRNA expressions of RANBP3L, OCN and RANKL detected by qRT-PCR. H, K and L： Protein expressions of OCN and RANKL detected by Western blotting. n=5. **P<0.01, ***P<0.001 vs Ctrl+Veh; #P<0.05, ##P<0.01, ###P<0.001 vs T2DM+Veh; ‡P<0.05, ‡‡P<0.01, ‡‡‡P<0.001 vs T2DM+KD+Veh.

图6 下调RANBP3L抑制TNF-α/NF-κB信号通路

Fig.6 Effects of RANBP3L knockdown and curcumin treatment on the TNF‑α/NF‑κB signaling pathway. A-E: Protein expressions of TNF-α, p-p65, p-IKKβ and p-IκB-α detected by Western blotting. n=5. ***P<0.001 vs Ctrl+Veh; #P<0.05, ##P<0.01 vs T2DM+Veh; ‡P<0.05, ‡‡‡P<0.001 vs T2DM+KD+Veh.

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