Chen Nan, Wang Na-Ni, Du Chen, Zhang Jia-Li, Guo Yi-Xun, Zhang Yan
Spine Disease Research Institute, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China; Key Laboratory of Theory and Therapy of Muscles and Bones, Ministry of Education, Shanghai 200032, China.
Department of Medicine, Zhejiang Academy of Traditional Chinese Medicine, Hangzhou 310007, China.
Phytomedicine. 2022 Apr;98:153982. doi: 10.1016/j.phymed.2022.153982. Epub 2022 Feb 7.
Our early studies performed on aged rats, ovariectomized (OVX) rats and diabetic mice, indicated the calciotropic role of Fructus Ligustri Lucidi (FLL), the fruit of Ligustrum lucidum Ait., in mediating calcium homeostasis which was partially attributed to its stimulation on renal calcium reabsorption.
This study aimed to explicate the underlying molecular mechanism and explore the potential bioactive ingredients in FLL.
The OVX C57BL/6 J mice were orally administered with low (FL, 75 mg/kg), middle (FM, 225 mg/kg) or high (FH, 675 mg/kg) dose of extract of Fructus Ligustri Lucidi for 10 weeks. The biological properties of trabecular bone were measured by micro-CT and H&E staining. The molecular expression was assessed by immunoblotting and immunostaining. The potential active components were identified by cell membrane chromatography (CMC) and explored in renal tubular cells with Fluo-3/AM fluorescent staining to indicate intracellular calcium level. The male mice fed with high calcium diet (1.2% Ca) and orally treated with active components for 3 weeks.
Treatment of OVX mice with FLL extract suppressed the elevation in urinary calcium level (FH, 0.081 ± 0.012, vs. OVX, 0.189 ± 0.038 mg/mg), and increased bone mineral density (FH, 62.41 ± 2.57, vs. OVX, 43.72 ± 8.43 mg/ccm) and percentage of trabecular bone area. It also decreased circulating PTH level (FH, 66.69 ± 10.94, vs. OVX, 303.50 ± 26.56 pg/ml) and up-regulated TRPV5 expression in renal cortex of OVX mice as well as enhanced the expression of PTH receptor (PTH1R) and the ratio of p-PKA/PKA. The PKA inhibitor H89 abolished the induction of serum, prepared from rats treated with FLL extract, on PKA/TRPV5 signaling in renal tubular cells. The CMC identified phenol glycosides, including salidroside and oleuropein, which increased intracellular calcium content, promoted expression of PTH1R and TRPV5 and ratio of p-PKA/PKA as well as decreased calcium excretion in urine of mice fed with high calcium diet.
Salidroside and oleuropein are major ingredients contributing to the anti-hypercalciuria effects of FLL via acting on PTH1R/PKA/TRPV5 signaling in kidney. Further translational research would be required.
我们早期对老年大鼠、去卵巢（OVX）大鼠和糖尿病小鼠进行的研究表明，女贞子果实女贞子在介导钙稳态中具有促钙作用，这部分归因于其对肾钙重吸收的刺激。
本研究旨在阐明其潜在的分子机制，并探索女贞子中的潜在生物活性成分。
对去卵巢的C57BL/6 J小鼠口服低剂量（FL，75 mg/kg）、中剂量（FM，225 mg/kg）或高剂量（FH，675 mg/kg）的女贞子提取物，持续10周。通过显微CT和苏木精-伊红染色测量小梁骨的生物学特性。通过免疫印迹和免疫染色评估分子表达。通过细胞膜色谱法（CMC）鉴定潜在的活性成分，并在肾小管细胞中用Fluo-3/AM荧光染色进行探索，以指示细胞内钙水平。给雄性小鼠喂食高钙饮食（1.2%钙），并口服活性成分3周。
用女贞子提取物治疗去卵巢小鼠可抑制尿钙水平的升高（FH，0.081±0.012，vs. OVX，0.189±0.038 mg/mg），并增加骨矿物质密度（FH，62.41±2.57，vs. OVX，43.72±8.43 mg/ccm）和小梁骨面积百分比。它还降低了循环中甲状旁腺激素水平（FH，66.69±10.94，vs. OVX，303.50±26.56 pg/ml），上调了去卵巢小鼠肾皮质中TRPV5的表达，并增强了甲状旁腺激素受体（PTH1R）的表达以及p-PKA/PKA的比值。PKA抑制剂H89消除了用女贞子提取物处理的大鼠血清对肾小管细胞中PKA/TRPV5信号通路的诱导作用。CMC鉴定出酚苷，包括红景天苷和橄榄苦苷，它们增加了细胞内钙含量，促进了PTH1R和TRPV5的表达以及p-PKA/PKA的比值，并降低了喂食高钙饮食小鼠的尿钙排泄。
红景天苷和橄榄苦苷是女贞子抗高钙尿作用的主要成分，它们通过作用于肾脏中的PTH1R/PKA/TRPV5信号通路发挥作用。需要进一步的转化研究。
