Hyperoside alleviates postmenopausal osteoporosis via regulating miR‐19a‐5p/IL‐17A axis

Postmenopausal osteoporosis (PMO) is a common osteoporosis. Hyperoside (Hyp), a natural flavonoid compound, has anti‐osteoporotic effects, but the underlying mechanisms remain poorly understood. Inflammatory cytokine IL‐17A is upregulated in PMO and plays vital roles in bone loss, but the upstream regulatory factors and mechanisms are still unknown.


INTRODUCTION
Osteoporosis is a common disorder in postmenopausal women. It is a highly prevalent disorder estimated to affect 200 million women and men worldwide, especially prevalent in older postmenopausal women, and increases the risk of fractures that can be associated with significant morbidity and mortality. 1 Postmenopausal imbalance between osteoclastic bone resorption and osteoblastic bone formation is the primary contributor to osteoporosis. Other important risk factors for postmenopausal osteoporosis (PMO) include postmenopause, old age, genetics, smoking, thinness, and many diseases and medications that impair bone health. 2 Although plenty studies have focused on PMO, its pathogenesis remains poorly understood.
IL-17A is a pro-inflammatory cytokine derived from activated T cells, presents with an elevated level in PMO serum and subsequently has been proved functional in augmenting osteoclast differentiation and bone resorption, leading to prominent bone loss. 3,4 In addition, anti-IL-17 antibody showed a better skeletal preservation effect on estrogen-deficient mice. 5 Increasing studies revealed that IL-17A plays crucial roles in the development and treatment of PMO, but the factors that regulate IL-17A expression in PMO are still unknown. 6 With the rapid development of sequencing technologies, up to 60% of proteincoding genes may be modulated by miRNAs, 7 which also have shown versatile functions in bone cell differentiation and function, bone development, and diseases. 8 Many studies have reported the critical role of miRNAs in PMO via regulating osteoblast differentiation. 9,10 However, the regulatory effect of miRNAs on IL-17A in PMO has not been fully investigated.
Currently, some anti-osteoporotic drugs have been widely used in the clinical treatment of PMO, including estrogen, Calcitonin, and bisphosphonate. However, due to the side effects such as gastrointestinal and renal complications, and long course of treatment, the clinical effect is unsatisfactory. 11 Therefore, it is urgently needed to develop more effective drugs in clinical practice. Traditional Chinese medicine (TCM) has been gradually revealed to be functional in PMO relief with few side effects. Hyperoside (Hyp) is an active flavonoid glycoside isolated from various medicinal plants, such as epimedium, hypericum, and perforatum, which are known to have anti-osteoporotic effects. A few studies have demonstrated that Hyp exerts comprehensive pharmacological functions, such as anti-inflammation, 12 anti-apoptosis, 13 and anti-infection. 14 Recently, Sun et al. found that Hyp showed protective effects on osteoarthritis mice both in vitro and in vivo. 15 However, whether this osteoprotective effect of Hyp is achieved by regulating IL-17A has not been reported.
In the present study, the expression of IL-17A in the peripheral blood of PMO patients was detected. Moreover, it is predicted by Tar-getScan and miRDB that miR-19a-5p might bind to IL-17A, and the regulatory effect of miR-19a-5p on IL-17A in PMO was investigated in receptor activator of nuclear factor-κB ligand (RANKL)-induced RAW264.7 osteoclasts. Moreover, the bone protection and regulatory effect of Hyp on miR-19a-5p/IL-17A axis were explored in ovariectomized (OVX) mice. Collectively, these results revealed that hyp/miR-19a-5p/IL-17A signal axis might act as a potential therapeutic strategy for PMO management.

Dual-luciferase reporter assay
The wild-type and mutant human IL-17A mRNA 3′UTR luciferase reporter vectors were constructed by amplifying human wild-type or TA B L E 1 List of PCR primer sequences used in mRNA expression analysis.

RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instruction. RNA was reversely tran-

Animals
All animal experiments were approved by the Institutional Animal Care and Use Committee of Shandong University of Traditional Chinese Medicine. Female C57BL/6 mice (weight 19-21 g) at 8-week-old were purchased from the Beijing Huafukang BioScience company (Beijing, China). The mice were bilaterally ovariectomized under anesthesia as described previously. 16 In the OVX group, the fallopian tubes and blood vessels were ligated and the ovaries were completely removed. In the sham operation group, adipose tissue with the same size as ovary was removed next to ovarian tissue.

Statistical analysis
All data are presented as the mean ± SD. Two-tailed Student's t-test was used for comparisons between two groups, and one-way analysis of variance (ANOVA) was used for multiple groups comparisons.
Correlations were analyzed by Pearson correlation. The diagnostic

IL-17A was upregulated in PMO patients and negatively correlated with miR-19a-5p
qRT-PCR result showed that IL-17A mRNA expression in the PBMCs of PMO patients was robustly increased as compared with the healthy control patients (HCs) group, p < .001 ( Figure 1A). Correspondingly, the IL-17A protein expression detected by ELISA was obviously upregulated in PMO patients' serum compared with the HCs group, p < .001 ( Figure 1B). Moreover, the protein expression of RANK and Cathepsin K, which are the typical osteoclast markers, in the PBMCs of PMO patients was also significantly increased as compared with that of HCs group, p < .001 ( Figure 1C).

IL-17A is the direct target gene of miR-19a-5p
To validate the post-transcriptional suppressive effect of miR-19a-5p on IL-17A, the potential complementary miR-19a-5p binding site on IL-17A 3′UTR was located by TargetScan software. Then dualluciferase reporter assay was used with co-transfection of miR-19a-5p mimics and plasmids encoding wild-type and mutant 3′UTR of IL-17A ( Figure 4A). The miR-19a-5p mimics markedly inhibited the activity of the luciferase encoded with wild-type IL-17A mRNA 3′UTR, p < .001 ( Figure 4B). Conversely, this suppressive effect of miR-19a-5p was not observed for the luciferase reporter activity of mutant IL-17A mRNA 3′UTR ( Figure 4C). These results suggested that IL-17A is the direct target of the miR-19a-5p by binding to the 3′UTR.

IL-17A increased while miR-19a-5p decreased in OVX mice
To further clarify the role of miR-19a-5p in vivo, the PMO mouse model was established by bilateral ovariectomy. As shown in Figure 5A, the microstructure of trabecular bone in OVX mice was irregularly arranged, and obviously sparse and thinner than that in the Control and Sham group. The mRNA and protein expression of IL-17A in OVX mice were significantly increased compared with Control and Sham group, p < .01 ( Figure 5B,C). Moreover, western blot results showed that the expression of RANK and Cathepsin K was also obviously elevated in OVX mice compared with the Control and Sham group, p < .001 ( Figure 5D). Notably, the miR-19a-5p expression of OVX mice femurs tissue was robustly decreased as compared with the other groups, p < .05 ( Figure 5E). These results proved that the change of miR-19a-

miR-19a-5p inhibited IL-17A expression and osteoclasts differentiation in OVX mice
Based on the above results, the miR-19a-5p mimics, inhibitor, or their NC vehicle were, respectively, injected via tail vein into the OVX mice.
As expected, the expression of miR-19a-5p was significantly increased in the OVX mice femurs tissue of miR-19a-5p mimics group (p < .0001), but reduced in miR-19a-5p inhibitor group (p < .05) compared with that in respective NC group ( Figure 6A). qRT-PCR result showed that the expression of IL-17A in the OVX mice femurs tissue was notably downregulated in miR-19a-5p mimics group (p < .05), while upregulated in miR-19a-5p inhibitor group (p < .01) compared with their NC group ( Figure 6B,C). Correspondingly, the expression of RANK and Cathepsin K was markedly decreased in miR-19a-5p mimics group (p < .001),

Hyp improved PMO by regulating miR-19a-5p/IL-17A axis in mice
To further explore the mechanism of Hyp in PMO, OVX mice were treated with Hyp at different doses. As shown in Figure 8A PMO pathogenesis is closely related to body immune dysfunction and systematic inflammation activation. 19 Recently, researches on IL-17A in bone metabolism further enhanced the connection between PMO and inflammation reaction. 20 29 Further, miR-19a-5p has been found to restrain the inflammatory response by downregulating the expression of TNF-α in macrophages. 30 In addition, miR-19a-5p showed suppressive effects on T cell proliferation and activation by inhibiting the expression of CD69. 31 Given the fact microRNAs always regulate a set of potential targets, whether miR-19a-5p could target inflammatory signaling or T cell function in PMO still needs further exploration.
Hyp is an active flavonoid glycoside and has anti-osteoporotic effects. It has been reported that Hyp and Icariin could improve the ovarian endocrine function through promoting the secretion of estrogen and progesterone. 32  In summary, the present study demonstrated that downregulation of miR-19a-5p was involved in osteoporosis induced by estrogen deficiency by enhancing IL-17A. Hyp presented a notable protective effect on PMO, and its related underlying molecular mechanism may involve miR-19a-5p/IL-17A axis, indicating that it is a potential therapeutic agent in the treatment of PMO. Targeted regulation of miR-19a-5p/IL-17A may be one of the key mechanisms of Hyp in the treatment of PMO, this mechanism enriches the theory of "deficiency of kidney essence" in TCM, and provides verification for TCM theory from a microscopic perspective. PMO is a very complex pathophysiological process, and its mechanism and influencing factors are diversified, complex, and cross process. The research perspective of this experiment is only part of it, and we will further study it.