吴茱萸碱抑制破骨细胞分化延缓骨丢失的研究

doi:10.3877/cma.j.issn.2095-1221.2022.02.004

目的

探讨吴茱萸碱对破骨细胞分化与骨吸收功能的调控及对骨质疏松症的治疗作用。

方法

取小鼠原代骨髓来源巨噬细胞分别给予0、10、20、50、100、200 μmol/L吴茱萸碱处理，CCK8检测细胞活力；然后利用原代骨髓来源巨噬细胞给予小鼠重组可溶性核因子κB受体活化因子配体与集落刺激因子行破骨细胞分化诱导，分别给予20与50 μmol/L吴茱萸碱干预。抗酒石酸酸性磷酸酶（TRAP）染色检测破骨细胞形成能力，荧光定量PCR分析破骨细胞分化相关基因表达，免疫荧光检测F肌动蛋白（F-actin）形成，扫描电镜观察破骨细胞骨吸收能力。7月龄C57BL/6小鼠灌胃给予100与200 mg/kg吴茱萸碱，给药3个月后Micro-CT检测小鼠骨密度与骨质量。采用单因素方差分析和t检验进行统计学分析。

结果

CCK8结果显示，与对照组相比，给予10、20、50、100 μmol/L吴茱萸碱处理后细胞活力无明显变化，差异无统计学意义（P > 0.05）；而给予200 μmol/L吴茱萸碱的细胞活力下降（100.64±0.18比47.54±5.58），差异具有统计学意义（P < 0.01）。与对照组相比，20 μmol/L吴茱萸碱的TRAP染色阳性细胞数[（200.57±28.35）比（142.29±19.21）个]、Trap （1.00±0.13比0.55±0.16）、组织蛋白酶K（Ctsk）（1.01±0.17比0.59±0.11）mRNA水平、骨吸收面积比（1.00±0.15比0.79±0.19）均减少，差异有统计学意义（P < 0.05）。与对照组相比，50 μmol/L吴茱萸碱的TRAP阳性细胞数[（200.57±28.35）比（112.71±12.18）个]、Trap （1.00±0.13比0.46±0.17）、Ctsk（1.01±0.17比0.49±0.12）、树突状细胞-特异性跨膜蛋白（DC- Stamp）（1.00±0.10比0.55±0.14）、c-Fos （1.01±0.10比0.58±0.14）、活化T细胞核因子c1 （Nfatc1）（1.00±0.10比0.59±0.14）、H+转运ATP酶v0亚基d2 （Atp6v0d2）的mRNA表达（1.00±0.10比0.59±0.18）、F-actin数量[（165.00± 18.50）比（98.33±21.15）个]和骨吸收面积比（1.00±0.15比0.62±0.10）均降低，差异有统计学意义（P < 0.05）。Micro-CT结果显示，与生理盐水组相比，100 mg/kg吴茱萸碱组小鼠骨密度有一定升高[（0.19±0.03）比（0.21±0.01）g/cm³]，但差异无统计学意义（P > 0.05）；与生理盐水组相比，200 mg/kg吴茱萸碱组小鼠胫骨的骨密度[（0.19±0.03）比（0.23±0.01）g/cm³]、骨体积比[（9.79±1.39）﹪比（11.62±1.18）﹪]、骨小梁数量[（2.43±0.29）比（3.08±0.43）/mm]上升，骨小梁分离度[（0.44±0.06）比（0.27±0.05）mm]下降，差异具有统计学意义（P < 0.05）。

结论

吴茱萸碱通过抑制破骨细胞分化与骨吸收功能延缓小鼠骨量丢失。

关键词: 吴茱萸碱, 破骨细胞分化, 骨吸收, 骨量

Objective

To investigate the regulation of evodiamine on osteoclast (OC) differentiation and bone resorption, and its therapeutic effect on osteoporosis.

Methods

The mouse primary bone marrow-derived macrophages were treated with 0, 10, 20, 50, 100, 200 μmol/L evodiamine respectively, and the cell viability was detected by CCK8. Primary bone marrow-derived macrophages were used to induce osteoclast differentiation in mice by administering recombinant soluble nuclear factor-κB receptor activator factor and colony-stimulating factor, and were intervened by 20 and 50 μmol/L evodiamine, respectively. The formation ability of osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining, the expression of osteoclast differentiation-related genes was analyzed by fluorescence quantitative PCR, the formation of F-actin was detected by immunofluorescence, and the bone resorption ability of osteoclasts was observed by scanning electron microscope. 7-month-old C57BL/6 mice were orally administrated with evodiamine daily at 100 or 200 mg/kg for 3 months. Bone density and bone quality of the mice were measured by Micro-CT. One-way ANOVA and t-test was used for statistical analysis.

Results

Compared with the control group, CCK8 results showed that there was no significant change in cell viability in the 10, 20, 50, and 100 μmol/L evodiamine-treated groups, and the difference was not statistically significant (P > 0.05) . 200 μmol/L evodiamine inhibited the cell viability (100.64±0.18 vs 47.54±5.58) , and the difference was statistically significant (P < 0.01) . 20 μmol/L evodiamine decreased the number of TRAP staining positive cells (200.57±28.35 vs 142.29±19.21) , the mRNA levels of Trap (1.00±0.13 vs 0.55±0.16) and cathepsin K (Ctsk) (1.01±0.17 vs 0.59±0.11) , the area of bone resorption (1.00±0.15 vs 0.79±0.19) , compared with the control group, and the difference was statistically significant (P < 0.05) . 50 μmol/L evodiamine reduced the number of TRAP staining positive cells (200.57±28.35 vs 112.71±12.18) , the mRNA levels of Trap (1.00±0.13 vs 0.46±0.17) , Ctsk (1.01±0.17 vs 0.49±0.12) , DC-Stamp (1.00±0.10 vs 0.55±0.14) , c-Fos (1.01±0.10 vs 0.58±0.14) , Nfatc1 (1.00±0.10 vs 0.59±0.14) and Atp6v0d2 (1.00±0.10 vs 0.59±0.18) , the number of F-actin (165.00±18.50 vs 98.33±21.15) and bone resorption area was also decreased (1.00±0.15 vs 0.62±0.10) , and the difference was statistically significant (P < 0.05) . Micro-CT results showed the bone density of the mice in the 100 mg/kg evodiamine group were somewhat elevated compared with control group [ (0.19±0.03) vs (0.21±0.01) g/cm³], but the difference was not statistically significant (P > 0.05) .The bone density [ (0.19±0.03) vs (0.23±0.01) g/cm³], bone volume ratio[ (9.79±1.39) ﹪ vs (11.62±1.18) ﹪], and the number of trabecular bones[ (2.43±0.29) vs (3.08±0.43) mm] in the evodiamine 200 mg/kg group were increased, and the separation of trabecular bones[ (0.44±0.06) vs (0.27±0.05) mm] was decreased, compared with the control group, and the difference was statistically significant (P < 0.05) .

Conclusion

Evodiamine inhibited bone loss by inhibiting osteoclast differentiation and bone resorption.

Key words: Evodiamine, Osteoclast differentiation, Bone resorption, Bone density

表1 引物序列信息

基因	引物序列（5’-3’）	预期扩增长度（bp）
β-Actin	上游AGGCAGCTAAATGCAGAGGGTACA	108
	下游AGCTTGCATCGATGGACACAGAGA
Trap	上游CTGGAGTGCACGATGCCAGCGACA	419
	下游TCCGTGCTCGGCGATGGACCAGA
Ctsk	上游AGGCAGCTAAATGCAGAGGGTACA	108
	下游AGCTTGCATCGATGGACACAGAGA
DC-Stamp	上游ACAAACAGTTCCAAAGCTTGC	74
	下游TCCTTGGGTTCCTTGCTTC
Nfatc1	上游CCGTTGCTTCCAGAAAATAACA	152
	下游TGTGGGATGTGAACTCGGAA
c-Fos	上游CCAGTCAAGAGCATCAGCAA	247
	下游AAGTAGTGCAGCCCGGAGTA
Atp6v0d2	上游AAGCCTTTGTTTGACGCTGT	199
	下游TTCGATGCCTCTGTGAGATG

表1 引物序列信息

基因	引物序列（5’-3’）	预期扩增长度（bp）
β-Actin	上游AGGCAGCTAAATGCAGAGGGTACA	108
	下游AGCTTGCATCGATGGACACAGAGA
Trap	上游CTGGAGTGCACGATGCCAGCGACA	419
	下游TCCGTGCTCGGCGATGGACCAGA
Ctsk	上游AGGCAGCTAAATGCAGAGGGTACA	108
	下游AGCTTGCATCGATGGACACAGAGA
DC-Stamp	上游ACAAACAGTTCCAAAGCTTGC	74
	下游TCCTTGGGTTCCTTGCTTC
Nfatc1	上游CCGTTGCTTCCAGAAAATAACA	152
	下游TGTGGGATGTGAACTCGGAA
c-Fos	上游CCAGTCAAGAGCATCAGCAA	247
	下游AAGTAGTGCAGCCCGGAGTA
Atp6v0d2	上游AAGCCTTTGTTTGACGCTGT	199
	下游TTCGATGCCTCTGTGAGATG

图1 骨髓巨噬细胞活力检测注：与0 μmol/L吴茱萸碱比较，^aP < 0.01，n = 3

图2 吴茱萸碱抑制破骨细胞分化（倒置荧光显微镜，×100）注：a ~ c图为破骨细胞TRAP染色，红色显示为阳性破骨细胞，对照组有大而多的破骨细胞，20 μmol/L吴茱萸碱的破骨细胞变小，数目减少，50 μmol/L吴茱萸碱仅有少量较小的破骨细胞；d图为破骨细胞数目统计，与0 μmol/L吴茱萸碱比较，^aP < 0.01，F = 31.767，n = 3

表2 吴茱萸碱对破骨细胞分化标记基因mRNA表达的影响（n = 3， ± s）

分组	Trap				DC-Stamp
分组	0 d	1 d	3 d	5 d	0 d	1 d	3 d	5 d
对照	1.00±0.17	1.34±0.30	9.16±1.54^a	20.50±2.65^a	1.01±0.09	0.90±0.10	12.08±2.48^a	31.03±1.80^a
吴茱萸碱	1.15±0.24	1.41±0.20	3.29±0.73^b	5.70±1.21^b	0.81±0.04^b	0.80±0.13	6.38±1.11^b	12.02±1.11^b
t值	0.867	0.334	5.977	8.799	3.765	1.098	3.631	15.542
P值	0.435	0.756	< 0.01	< 0.01	< 0.05	0.334	< 0.05	< 0.01
分组	Ctsk				c-Fos
分组	0 d	1 d	3 d	5 d	0 d	1 d	3 d	5 d
对照	1.00±0.10	1.27±0.30	11.12±1.67^a	30.25±4.50^a	0.99±0.20	9.78±1.39^a	6.19±1.67^a	2.57±0.88
吴茱萸碱	1.21±0.35	1.67±0.29	5.27±1.24^b	16.92±2.70^b	1.21±0.31	5.46±1.14^b	2.83±1.12^b	1.76±0.32
t值	1.012	1.653	4.869	4.396	1.026	4.164	2.884	1.509
P值	0.369	0.174	< 0.01	< 0.05	0.363	< 0.05	< 0.05	0.206

表2 吴茱萸碱对破骨细胞分化标记基因mRNA表达的影响（n = 3， ± s）

分组	Trap				DC-Stamp
分组	0 d	1 d	3 d	5 d	0 d	1 d	3 d	5 d
对照	1.00±0.17	1.34±0.30	9.16±1.54^a	20.50±2.65^a	1.01±0.09	0.90±0.10	12.08±2.48^a	31.03±1.80^a
吴茱萸碱	1.15±0.24	1.41±0.20	3.29±0.73^b	5.70±1.21^b	0.81±0.04^b	0.80±0.13	6.38±1.11^b	12.02±1.11^b
t值	0.867	0.334	5.977	8.799	3.765	1.098	3.631	15.542
P值	0.435	0.756	< 0.01	< 0.01	< 0.05	0.334	< 0.05	< 0.01
分组	Ctsk				c-Fos
分组	0 d	1 d	3 d	5 d	0 d	1 d	3 d	5 d
对照	1.00±0.10	1.27±0.30	11.12±1.67^a	30.25±4.50^a	0.99±0.20	9.78±1.39^a	6.19±1.67^a	2.57±0.88
吴茱萸碱	1.21±0.35	1.67±0.29	5.27±1.24^b	16.92±2.70^b	1.21±0.31	5.46±1.14^b	2.83±1.12^b	1.76±0.32
t值	1.012	1.653	4.869	4.396	1.026	4.164	2.884	1.509
P值	0.369	0.174	< 0.01	< 0.05	0.363	< 0.05	< 0.05	0.206

表3 不同剂量吴茱萸碱对破骨细胞分化标记基因mRNA表达的影响（ ± s）

分组	实验次数	Trap	Ctsk	DC-Stamp	c-Fos	Nfatc1	Atp6v0d2
对照	3	1.00±0.13	1.01±0.17	1.00±0.10	1.01±0.10	1.00±0.10	1.00±0.10
20 μmol/L吴茱萸碱	3	0.55±0.16^a	0.59±0.11^a	0.81±0.04	0.95±0.18	0.73±0.16	0.83±0.09
50 μmol/L吴茱萸碱	3	0.46±0.17^a	0.49±0.12^a	0.55±0.14^a	0.58±0.14^a	0.59±0.14^a	0.59±0.18^a
F值		11.015	12.591	14.920	7.940	7.462	7.731
P值		< 0.01	< 0.01	< 0.01	< 0.05	< 0.05	< 0.05

表3 不同剂量吴茱萸碱对破骨细胞分化标记基因mRNA表达的影响（ ± s）

分组	实验次数	Trap	Ctsk	DC-Stamp	c-Fos	Nfatc1	Atp6v0d2
对照	3	1.00±0.13	1.01±0.17	1.00±0.10	1.01±0.10	1.00±0.10	1.00±0.10
20 μmol/L吴茱萸碱	3	0.55±0.16^a	0.59±0.11^a	0.81±0.04	0.95±0.18	0.73±0.16	0.83±0.09
50 μmol/L吴茱萸碱	3	0.46±0.17^a	0.49±0.12^a	0.55±0.14^a	0.58±0.14^a	0.59±0.14^a	0.59±0.18^a
F值		11.015	12.591	14.920	7.940	7.462	7.731
P值		< 0.01	< 0.01	< 0.01	< 0.05	< 0.05	< 0.05

图3 吴茱萸碱抑制F-actin形成与骨吸收（共聚焦荧光显微镜，×100；电镜扫描，×400）注：a ~ c图为免疫荧光染色，绿色显示为F-actin阳性表达，蓝色为DAPI染色，显示细胞核，对照组F-actin数量多（a图），20 μmol/L吴茱萸碱的F-actin数量减少（b图），50 μmol/L吴茱萸碱的F-actin数量显著减少（c图）；d图为免疫荧光染色统计，n = 3；e ~ g图为电镜扫描，显示骨吸收所形成的陷窝面积；对照组骨吸收陷窝面积较大（e图），吴茱萸碱20 μmol/L组陷窝面积减少（f图），吴茱萸碱50 μmol/L组陷窝面积显著减少（g图）；h图为电镜扫描统计，n = 3；与吴茱萸碱0 μmol/L组比，^aP < 0.05

图4 Micro-CT扫描胫骨组织注：灰色多代表骨质多；a、e图为7月龄小鼠有多而密的骨质，骨量较高；b、f图为生理盐水组骨质大量丢失，骨量低；c、g图为100 mg/kg吴茱萸碱组骨质有一定的增多；d、h图为200 mg/kg吴茱萸碱组骨质显著增多

表4 Micro-CT扫描参数统计（ ± s）

分组	动物数	骨密度（g/cm³）	骨体积比（﹪）	骨小梁数量（/mm）	骨小梁厚度（μm）	骨小梁分离度（mm）
7月龄	8	0.24±0.03	15.43±1.46	3.73±0.51	39.08±4.40	0.22±0.04
生理盐水	8	0.19±0.03^a	9.79±1.39^a	2.43±0.29^a	33.11±3.21^a	0.44±0.06^a
100 mg/kg吴茱萸碱	8	0.21±0.01	8.50±0.99	2.50±0.33	33.76±3.21	0.44±0.06
200 mg/kg吴茱萸碱	8	0.23±0.01^b	11.62±1.18^b	3.08±0.43^b	35.91±2.48	0.27±0.05^b
F值		3.972	10.215	6.163	1.836	18.915
P值		< 0.05	< 0.01	< 0.05	> 0.05	< 0.01

表4 Micro-CT扫描参数统计（ ± s）

分组	动物数	骨密度（g/cm³）	骨体积比（﹪）	骨小梁数量（/mm）	骨小梁厚度（μm）	骨小梁分离度（mm）
7月龄	8	0.24±0.03	15.43±1.46	3.73±0.51	39.08±4.40	0.22±0.04
生理盐水	8	0.19±0.03^a	9.79±1.39^a	2.43±0.29^a	33.11±3.21^a	0.44±0.06^a
100 mg/kg吴茱萸碱	8	0.21±0.01	8.50±0.99	2.50±0.33	33.76±3.21	0.44±0.06
200 mg/kg吴茱萸碱	8	0.23±0.01^b	11.62±1.18^b	3.08±0.43^b	35.91±2.48	0.27±0.05^b
F值		3.972	10.215	6.163	1.836	18.915
P值		< 0.05	< 0.01	< 0.05	> 0.05	< 0.01

图5 光学显微镜下观察胫骨组织（TRAP染色，×400）注：红色为TRAP阳性，显示活性的破骨细胞，红色多代表破骨细胞活性高；a图为7月龄组仅有少量的TRAP阳性着色；b图为生理盐水组有大面积TRAP阳性着色，提示破骨细胞活性高；c图为100 mg/kg吴茱萸碱组TRAP阳性量低于生理盐水组；d图为200mg/kg吴茱萸碱组TRAP阳性量显著低于生理盐水组

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Evodiamine regulates osteoclast differentiation and inhibits bone loss

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Evodiamine regulates osteoclast differentiation and inhibits bone loss