Naringin inhibits iron deposition and cell apoptosis in bone tissue of osteoporotic rats

doi:10.12307/2024.593

Abstract

Abstract: BACKGROUND: It has been found that abnormal apoptosis of bone tissue cells induced by abnormal iron metabolism plays an important role in the progression of osteoporosis.
OBJECTIVE: To investigate the effect of naringin on iron metabolism and cell apoptosis in bone tissue of rats with osteoporosis.
METHODS: Fifty 2-month-old female Sprague-Dawley rats were randomly divided into five groups with 10 rats in each group: sham group, osteoporosis group, naringin low-dose group, naringin high-dose group, and naringin high-dose+DKK-1 group. Except for the sham group, rat models of osteoporosis were established by removing bilateral ovarian tissues in the other groups. At 8 weeks after modeling, rats in the naringin low- and high-dose groups were given 100 and 400 mg/kg/d naringenin by gavage, respectively, and rats in the naringenin high dose+DKK-1 group were given 400 mg/kg/d naringin by gavage and subcutaneous injection of 25 mg/kg/d DKK-1, an inhibitor of the Wnt1 signaling pathway, for 7 consecutive days. Relevant indexes were detected after administration.
RESULTS AND CONCLUSION: Compared with the osteoporosis group, naringin could enhance the bone mineral density and serum calcium and superoxide dismutase levels in rats (P < 0.05), and reduce the serum levels of osteocalcin, malondialdehyde, and phosphorus (P < 0.05), while DKK-1 could partially inhibit the interventional effect of naringin (P < 0.05). Results from Micro-CT scanning, hematoxylin-eosin and TUNEL staining showed that compared with the osteoporosis group, naringin significantly improved bone microstructure and reduced the rate of cell apoptosis, while DKK-1 partially inhibited the interventional effect of naringin. Immunofluorescence staining results showed that compared with the osteoporosis group, naringin could reduce the oxygen content, anti-tartaric acid phosphatase expression, and elevate the expression of alkaline phosphatase in active tibia tissues (P < 0.05), while DKK-1 could partially inhibit the interventional effect of naringin (P < 0.05). Results from Prussian blue staining and immunohistochemical staining showed that compared with the osteoporosis group, naringin reduced iron deposition in bone and liver tissues as well as the expression of transferrin receptor 1 (P < 0.05), and elevated the protein expression of ferroportin 1 (P < 0.05) in bone tissue, and DKK-1 partially inhibited the intervention of naringin (P < 0.05). PCR and western blot assay of tibia specimens showed that compared with the osteoporosis group, naringin decreased the expression of anti-tartrate acid phosphatase, transferrin receptor 1 and Bax (P < 0.05), and elevated the expression of alkaline phosphatase, ferroportin 1, Bcl-2, Wnt1 and β-catenin (P < 0.05), while DKK-1 partially inhibited the interfering effect of naringin (P < 0.05). To conclude, naringin inhibits the progression of osteoporosis by reducing iron deposition and apoptosis rate in bone tissue, which may be related to the activation of the Wnt1 signaling pathway.

Key words: naringin, osteoporosis, iron metabolism, Wnt1 signaling pathway, cell apoptosis

CLC Number:

Lan Shuangli, Xiang Feifan, Deng Guanghui, Xiao Yukun, Yang Yunkang, Liang Jie. Naringin inhibits iron deposition and cell apoptosis in bone tissue of osteoporotic rats [J]. Chinese Journal of Tissue Engineering Research, 2025, 29(5): 888-898.

Figures/Tables 11

2.1 各组大鼠造模结果及一般表现在实验过程中未发生动物死亡的现象。造模后，假手术组大鼠活泼活动，反应机敏，皮毛顺滑浓密，摄食、饮水及睡眠未见异常；骨质疏松组大鼠精神状态偏于狂躁不安，反应迟钝，皮毛暗淡稀疏，大鼠在实验过程中往往处于少动嗜睡的状态，摄食与饮水意愿不强烈；柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组大鼠的情绪、精神状态较为稳定，反应较为敏捷，皮毛较为顺整。与假手术组相比，骨质疏松组大鼠血清钙水平和骨密度明显下降(P < 0.05)，血清骨钙素和磷的水平明显升高(P < 0.05)，证实造模成功。 2.2 各组大鼠血清中骨钙素、钙、磷水平及骨密度的变化与假手术组相比，骨质疏松组、柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组大鼠血清钙水平和骨密度下降(P < 0.05)，骨钙素和磷水平升高(P < 0.05)；与骨质疏松组相比，柚皮苷干预组大鼠血清钙水平和骨密度升高(P < 0.05)，骨钙素和磷水平下降(P < 0.05)；与柚皮苷高剂量组相比，柚皮苷低剂量组、柚皮苷高剂量+DKK-1组大鼠血清钙水平和骨密度下降(P < 0.05)，骨钙素和磷水平升高(P < 0.05)，见图1。 2.3 各组大鼠骨微结构的变化 Micro-CT扫描结果显示，与假手术组相比，骨质疏松组、柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组大鼠胫骨组织的平均骨体积分数和平均骨小梁数下降(P < 0.05)，骨小梁间距升高(P < 0.05)；与骨质疏松组相比，柚皮苷干预组大鼠胫骨组织的平均骨体积分数和平均骨小梁数升高(P < 0.05)，骨小梁间距下降(P < 0.05)；与柚皮苷高剂量组相比，柚皮苷低剂量组、柚皮苷高剂量+DKK-1组大鼠胫骨组织的平均骨体积分数和平均骨小梁数下降(P < 0.05)，骨小梁间距升高(P < 0.05)，见图2。 2.4 各组大鼠骨组织病理损伤苏木精-伊红染色结果显示，假手术组大鼠胫骨组织形态正常，结构完整，骨小梁粗细匀称，排列致密有序，骨小梁内纹理清晰可辨，骨皮质厚度均一，未见明显异常；骨质疏松组大鼠胫骨组织结构破坏明显，骨小梁数量减少，排列紊乱，结构变细，模糊不清晰，部分有断裂、缺失现象，髓腔扩张明显，骨皮质变薄，出现部分空洞结构；与骨质疏松组相比，柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组大鼠胫骨组织病理症状明显改善，骨小梁结构宽度接近正常，排列较为规则，骨皮质明显增厚，尤以柚皮苷低剂量组、柚皮苷高剂量组改善程度最为明显，见图3。 2.5 各组大鼠胫骨组织细胞凋亡情况胫骨组织TUNEL染色显示，与假手术组相比，骨质疏松组、柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组细胞凋亡率升高(P < 0.05)；与骨质疏松组相比，柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组细胞凋亡率下降(P < 0.05)；与柚皮苷高剂量组相比，柚皮苷低剂量组、柚皮苷高剂量+DKK-1组细胞凋亡率升高(P < 0.05)，见图4。 2.6 各组大鼠胫骨组织中相关基因的表达胫骨组织PCR检测显示，与假手术组相比，骨质疏松组、柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组抗酒石酸酸性磷酸酶、铁吸收相关蛋白受体1、Bax的mRNA表达升高(P < 0.05)，碱性磷酸酶、膜铁转运蛋白1、Bcl-2、Wnt1、β-catenin的mRNA表达降低(P < 0.05)；与骨质疏松组相比，柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组抗酒石酸酸性磷酸酶、铁吸收相关蛋白受体1、Bax的mRNA表达降低(P < 0.05)，碱性磷酸酶、膜铁转运蛋白1、Bcl-2、Wnt1、β-catenin的mRNA表达升高(P < 0.05)；与柚皮苷高剂量组相比，柚皮苷低剂量组、柚皮苷高剂量+DKK-1组抗酒石酸酸性磷酸酶、铁吸收相关蛋白受体1、Bax的mRNA表达升高(P < 0.05)，碱性磷酸酶、膜铁转运蛋白1、Bcl-2、Wnt1、β-catenin的mRNA表达降低(P < 0.05)，见图5。 2.7 各组大鼠胫骨组织中氧化应激水平胫骨组织超氧化物阴离子二氢乙啶染色显示，与假手术组相比，骨质疏松组、柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组大鼠活性氧含量升高(P < 0.05)；与骨质疏松组相比，柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组活性氧含量降低(P < 0.05)；与柚皮苷高剂量组相比，柚皮苷低剂量组、柚皮苷高剂量+DKK-1组活性氧含量升高(P < 0.05)，见图6。与假手术组相比，骨质疏松组、柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组大鼠血清丙二醛水平升高(P < 0.05)，超氧化物歧化酶水平下降(P < 0.05)；与骨质疏松组相比，柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组大鼠血清丙二醛水平下降(P < 0.05)，超氧化物歧化酶水平升高(P < 0.05)；与柚皮苷高剂量组相比，柚皮苷低剂量组、柚皮苷高剂量+DKK-1组大鼠血清丙二醛水平明显升高(P < 0.05)，超氧化物歧化酶水平下降(P < 0.05)，见图7。 2.8 各组大鼠胫骨组织和肝组织中铁沉积情况普鲁士蓝染色显示，与假手术组相比，骨质疏松组、柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组大鼠肝组织与骨组织中普鲁士蓝阳性细胞比例升高(P < 0.05)；与骨质疏松组相比，柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组大鼠肝组织与骨组织中中普鲁士蓝阳性细胞的比例降低(P < 0.05)；与柚皮苷高剂量组相比，柚皮苷低剂量组、柚皮苷高剂量+DKK-1组大鼠肝组织与骨组织中普鲁士蓝阳性细胞比例升高(P < 0.05)，见图8。胫骨组织免疫组化染色显示，与假手术组相比，骨质疏松组、柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组膜铁转运蛋白1的表达降低(P < 0.05)，铁吸收相关蛋白受体1的表达升高(P < 0.05)；与骨质疏松组相比，柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组膜铁转运蛋白1的表达升高(P < 0.05)，铁吸收相关蛋白受体1的表达降低(P < 0.05)；与柚皮苷高剂量组相比，柚皮苷低剂量组、柚皮苷高剂量+DKK-1组膜铁转运蛋白1的表达降低(P < 0.05)，铁吸收相关蛋白受体1的表达升高(P < 0.05)，见图9。 2.9 各组大鼠胫骨组织中抗酒石酸酸性磷酸酶和碱性磷酸酶的表达胫骨组织免疫荧光染色显示，与假手术组相比，骨质疏松组、柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组碱性磷酸酶表达明显降低(P < 0.05)，抗酒石酸酸性磷酸酶表达升高(P < 0.05)；与骨质疏松组相比，柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组碱性磷酸酶表达升高(P < 0.05)，抗酒石酸酸性磷酸酶表达明显降低(P < 0.05)；与柚皮苷高剂量组相比，柚皮苷低剂量组、柚皮苷高剂量+DKK-1组碱性磷酸酶表达降低(P < 0.05)，抗酒石酸酸性磷酸酶表达升高(P < 0.05)，见图10。 2.10 各组大鼠胫骨组织中Wnt1信号通路相关蛋白的表达胫骨组织Western blot检测显示，与假手术组相比，骨质疏松组、柚皮苷低剂量组、柚皮苷高剂量组、柚皮苷高剂量+DKK-1组Bcl-2、Wnt1、β-catenin蛋白表达降"

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