Amelioration of Fructus Ligustri Lucidi and its phenol glycosides on hypercalciuria via stimulating PTH1R/PKA/TRPV5 signaling

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.