Wenshen Tongluo Zhitong Recipe Improves the Osteogenic Differentiation of BMSC and Bone Loss in Senile Osteoporosis Model Mice by Inhibiting Macrophage Senescence
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摘要:
目的 探讨温肾通络止痛方调控巨噬细胞衰老对老年性骨质疏松症(Senile osteoporosis, SOP)的干预作用。 方法 利用过氧化氢制备衰老巨噬细胞模型并随机分为对照组、模型组、含药血清低剂量组、含药血清高剂量组。β-半乳糖苷酶染色以及qPCR、Western blot检测衰老指标p21和p53的mRNA及蛋白表达水平, 探讨含药血清对巨噬细胞衰老的影响。使用活性氧(Reactive oxygen species, ROS)染色和线粒体膜电位检测试剂盒(JC-1)检测巨噬细胞线粒体功能。qPCR检测巨噬细胞极化相关分子白细胞介素-6(Interleukin-6, IL-6)、白细胞介素-10(Interleukin-10, IL-10)、CD206、一氧化氮合酶(Inducible nitric oxide synthase, iNOS)的mRNA水平, 免疫荧光检测巨噬细胞极化相关分子精氨酸酶(Arginase, ARG1)和iNOS的蛋白表达, 评估含药血清对巨噬细胞极化的影响。qPCR检测成骨相关指标骨钙蛋白(Osteocalcin, OCN)、Ⅰ型胶原蛋白(Collagen type Ⅰ α 1, Col1a1)、Runt相关转录因子2(Runt-related transcription factor 2, Runx2)的mRNA水平以及ALP染色和茜素红染色, 评估含药血清处理的巨噬细胞条件培养基对骨髓间充质干细胞(Bone marrow mesenchymal stem cell, BMSC)成骨分化的影响。将C57BL/6J小鼠随机分为对照组、模型组、温肾通络止痛方低剂量组、温肾通络止痛方高剂量组, D-半乳糖制备SOP小鼠模型。Micro-CT分析小鼠股骨微结构, HE染色检测小鼠股骨病理变化, Western blot检测小鼠胫骨衰老相关分子p21、p53和成骨相关指标OCN、Runx2蛋白表达水平, qPCR检测小鼠胫骨衰老相关分子p21、p53 mRNA和巨噬细胞极化相关分子IL-6、iNOS、CD206、IL-10 mRNA的表达水平, 评估温肾通络止痛方对小鼠SOP模型的影响。 结果 与模型组相比, 温肾通络止痛方含药血清干预后, 衰老阳性细胞数量明显减少, p21和p53 mRNA和蛋白表达均显著降低(P＜0.05, P＜0.01, P＜0.001)。此外, 含药血清可显著抑制H2O2诱导的巨噬细胞ROS的产生(P＜0.05, P＜0.001)、线粒体膜电位下降(P＜0.05)。含药血清处理后, 巨噬细胞M1相关基因iNOS的mRNA表达下调(P＜0.01), M2相关基因CD163和CD206 mRNA表达上调(P＜0.05), iNOS荧光强度显著降低(P＜0.01), ARG1的荧光强度显著增加(P＜0.05)。含药血清处理的巨噬细胞条件培养基可增加ALP阳性细胞数(P＜0.01, P＜0.001)、茜素红阳性面积(P＜0.05), 以及升高成骨相关基因Runx2、Col1a1和OCN mRNA表达(P＜0.05, P＜0.01)。Micro-CT结果显示, 与模型组相比, 温肾通络止痛方组小鼠股骨BMD、BV/TV、Tb.N显著增加(P＜0.05, P＜0.01, P＜0.001), Tb.Sp显著降低(P＜0.05, P＜0.01);HE结果显示, 骨小梁明显改善, 数量增多变宽。此外, 温肾通络止痛方可显著抑制D-半乳糖诱导的小鼠胫骨衰老相关基因p21和p53 mRNA和蛋白上调(P＜0.05, P＜0.001, P＜0.000 1), 促进成骨相关指标OCN、Runx2蛋白表达(P＜0.01, P＜0.000 1), 促进M1相关基因IL-6和iNOS下调(P＜0.05), 同时可促进M2相关基因IL-10和CD206表达(P＜0.05, P＜0.01)。 结论 温肾通络止痛方可能通过抑制巨噬细胞衰老, 促进BMSC成骨分化, 从而发挥抗骨质疏松效应。 Abstract:OBJECTIVE To study the intervention effects of Wenshen Tongluo Zhitong Recipe(WTZR) on macrophage senescence and senile osteoporosis. METHODS The senescent macrophage model was established using hydrogen peroxide (H2O2) and subsequently divided into four groups: control, model, low-dose drug-treated serum, and high-dose drug-treated serum.β-galactosidase staining, Western blot and qPCR were employed to evaluate the mRNA expression of senescence markers p21 and p53. ROS staining and JC-1 staining were applied to assess mitochondrial function in macrophages. The mRNA levels of Interleukin(IL)-6, IL-10, CD206, and inducible nitric oxide synthase (iNOS) were determined by qPCR analysis. Immunofluorescence was used to evaluate arginase(ARG1) and iNOS protein expressions for assessing the impact of drug-containing serum on macrophage polarization. qPCR analysis was conducted to measure osteocalcin(OCN), collagen type Ⅰ alpha 1(Col1a1), runt-related transcription factor 2(Runx2) mRNA levels as osteoblast-related markers; ALP staining along with alizarin red staining were performed to evaluate the effect of macrophage conditioned medium treated with drug-containing serum on bone marrow mesenchymal stromal cell(BMSC)osteogenic differentiation. C57BL/6J mice were randomly allocated into four groups: control group, model group, WTZR low-dose group, and WTZR high-dose group. The senile osteoporosis (SOP) mouse model was established by D-galactose. Micro-CT scanning analyzed femur microstructure while HE staining detected pathological changes in femur bone tissue samples collected from each experimental condition. Furthermore, Western blot was used to detect the senescence-related molecules p21 and p53 and the osteogenesis-related markers OCN and Runx2, qPCR analysis measured tibial expression levels of senescence-related molecules (p21, p53) as well as macrophage polarisation-related molecules (IL-6, iNOS, CD206, and IL-10) to assess the effect of this compound on a mouse model simulating SOP. RESULTS Following intervention with serum containing WTZR, there was a significant decrease in the number of senescent positive cells compared to the model group. Additionally, there was a notable decrease in p21 and p53 mRNA and proteins expression (P < 0.05, P < 0.01, P < 0.001). Furthermore, drug-containing WTZR effectively inhibited ROS production induced by H2O2 and mitigated mitochondrial membrane potential reduction in macrophages (P < 0.05, P < 0.001). Treatment with drug-containing WTZR resulted in down-regulated mRNA expression of M1-related gene iNOS (P < 0.05) while up-regulating mRNA expression of M2-related genes CD163 and CD206 (P < 0.05). The drug-containing WTZR significantly reduced fluorescence intensity for iNOS (P < 0.01) while increasing ARG1 (P < 0.05) fluorescence intensity. Moreover, conditioned medium from macrophages treated with drug-containing serum increased ALP positive cell count (P < 0.01, P < 0.001), alizarin red positive area (P < 0.05), as well as Col1a1, Runx2 and OCN mRNA expression levels (P < 0.05, P < 0.01). The Tb.N, BMD, and BV/TV were significantly higher in the WTZR group compared to the model group (P < 0.05, P < 0.01, P < 0.001); meanwhile, Tb.Sp was notably lower than that observed in the model group (P < 0.05, P < 0.01); bone trabeculae were significantly improved, increased in number and widened. Additionally, the compound could significantly inhibit the D-galactose induced up-regulation of tibial senescence-related genes and proteins p21 and p53(P < 0.05, P < 0.001, P < 0.0001), promote the expression of osteogenesis-related markers OCN and Runx2 protein(P < 0.01, P < 0.0001), promote the down-regulation of M1 related genes IL-6 and iNOS (P < 0.05), and promote the expression of M2 related genes IL-10 and CD206 (P < 0.05, P < 0.01). CONCLUSION Wenshen Tongluo Zhitong Recipe may play an anti-osteoporosis effect by inhibiting macrophage senescence and promoting the osteogenic differentiation of BMSC. -
图 1 巨噬细胞衰老模型的建立
注: A.β-半乳糖苷酶染色检测造模后巨噬细胞衰老情况, 红色三角形指示绿色的衰老阳性细胞; B.CCK-8检测造模后巨噬细胞的细胞活力; 与对照组比较, *P＜0.05, ***P＜0.001。x±s, n=3。
Figure 1. Establishment of senescent macrophage model
图 2 含药血清对巨噬细胞衰老的影响
注: A.β-半乳糖苷酶染色检测含药血清给药后巨噬细胞衰老情况, 红色三角形指示绿色的衰老阳性细胞; B.qPCR检测含药血清干预后巨噬细胞p21、p53 mRNA的表达；C.Western blot检测含药血清干预后巨噬细胞p21、p53蛋白的表达; 与模型组比较, *P＜0.05, **P＜0.01, ***P＜0.001。x±s, n=3。
Figure 2. Effect of drug-containing serum on macrophage senescence
图 3 含药血清对衰老巨噬细胞线粒体功能的影响
注: A.ROS染色检测含药血清给药后衰老巨噬细胞ROS水平; B.JC-1检测含药血清给药后衰老巨噬细胞线粒体功能；与模型组比较, *P＜0.05, ***P＜0.001。x±s, n=3。
Figure 3. Effect of drug-containing serum on mitochondrial function in senescent macrophages
图 4 含药血清对衰老巨噬细胞极化的影响
注: A.qPCR检测含药血清干预后巨噬细胞iNOS、CD206、CD163 mRNA的表达水平; B.免疫荧光检测含药血清给药后衰老巨噬细胞iNOS与ARG1的表达水平；与模型组比较, *P＜0.05, **P＜0.01, ***P＜0.001。x±s, n=3。
Figure 4. Effect of drug-containing serum on the polarization of senescent macrophages
图 5 含药血清处理的巨噬细胞条件培养基对BMSC成骨分化的影响
注: A.ALP染色、茜素红染色检测含药血清处理的巨噬细胞条件培养基对BMSC成骨的影响; B.qPCR检测含药血清处理的巨噬细胞条件培养基对BMSC成骨相关基因Col1a1、Runx2和OCN mRNA表达; 与模型组-CM比较, *P＜0.05, **P＜0.01, ***P＜0.001。x±s, n=3。
Figure 5. Effect of conditioned medium derived from macrophage treated with drug-containing serum on osteogenic differentiation of BMSC
图 6 温肾通络止痛方对小鼠股骨骨微结构的影响
注: A.Micro-CT检测各组小鼠股骨微结构; B.各组小鼠BMD、Tb.Sp、BV/TV、Tb.N比较。
与模型组比较, *P＜0.05, **P＜0.01, ***P＜0.001，****P＜0.000 1。x±s, n=6。Figure 6. Effect of Wenshen Tongluo Zhitong Recipe on femoral bone microstructure in mice
图 7 温肾通络止痛方对小鼠股骨病理变化的影响
Figure 7. Effect of Wenshen Tongluo Zhitong Recipe on pathological changes of femur in mice
图 8 温肾通络止痛方对小鼠胫骨衰老和巨噬细胞极化相关基因的影响
注: A.qPCR检测温肾通络止痛方干预后小鼠胫骨p21、p53 mRNA的表达水平; B.qPCR检测温肾通络止痛方干预后小鼠胫骨IL-6、iNOS、IL-10和CD206 mRNA的表达水平; C.Western blot检测温肾通络止痛方干预后小鼠胫骨p21、p53、OCN和Runx2蛋白的表达水平；与模型组比较, *P＜0.05, **P＜0.01, ***P＜0.001, ****P＜0.000 1。x±s, n=6。
Figure 8. Effects of Wenshen Tongluo Zhtong Recipe on genes related to tibial aging and macrophage polarization in mice
表 1 qPCR引物序列
Table 1. qPCR Primer sequence
基因 引物序列(5'→3') 引物长度/bp GAPDH F: ACGGCAAATTCAACGGCACAG 21 R: ACACCAGTAGACTCCACGACATAC IL-6 F: CTCCCAACAGACCTGTCTATAC 22 R: CCATTGCACAACTCTTTTCTCA IL-10 F: TTCTTTCAAACAAAGGACCAGC 22 R: GCAACCCAAGTAACCCTTAAAG CD206 F: CCTATGAAAATTGGGCTTACGG 22 R: CTGACAAATCCAGTTGTTGAGG iNOS F: ATCTTGGAGCGAGTTGTGGATTGTC 25 R: TAGGTGAGGGCTTGGCTGAGTG Col1a1 F: TGAACGTGGTGTACAAGGTC 20 R: CCATCTTTACCAGGAGAACCA OCN F: AGCAGGAGGGCAATAAGGTAGTG 23 R: CTCGTCACAAGCAGGGTTAAGC p21 F: ATGTCCAATCCTGGTGATGTC 21 R: GAAGTCAAAGTTCCACCGTTC p53 F: TGGAAGGAAATTTGTATCCCGA 22 R: GTGGATGGTGGTATACTCAGAG 
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