Background
Osteoporosis is defined as a systemic skeletal disease that is characterized by low bone mass and micro-architectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture[
1]. According to the World Health Organization[
2], osteoporosis occurs when the bone mineral density falls more than 2.5 standard deviations (SD) below the standard reference for maximum bone mineral density of young adult females. The bone mass in females begins to decline slowly after the age of 35 to 40, followed by a phase of dramatic bone loss after menopause. This is due to estrogen deficiency which occurs naturally with aging or by surgical ovariectomy.
By 50 years of age, the bone mass in women is only two-thirds of that in men[
3]. The relatively lower bone mass in women is due to a combination of lower peak bone mass and faster rate of bone loss. This leads to higher incidence of osteoporosis in elderly women compared to men[
4]. Osteoporosis should be managed appropriately as statistics have shown that 1 in 3 women aged more than 50 years old suffered an osteoporotic fracture during their lifetime[
5].
Estrogen Replacement Therapy (ERT) is one of the main form of treatment and prevention of postmenopausal osteoporosis. Estrogen given alone or in combination with progesterone is able to prevent postmenopausal osteoporosis effectively[
6]. Estrogen binds to its receptors on the osteoclast surface, causing the release of chemical mediators which led to reduction of osteoclastic activity and inhibition of bone resorption[
7].
The “Women’s Health Initiative Study” found that women who took ERT have slightly higher rates of ovarian cancer, breast cancer, heart attack, thromboembolism, stroke, and Alzheimer’s disease[
8‐
10]. These negative reports on the serious adverse-effects of ERT have led to many postmenopausal women searching for available alternatives for their postmenopausal symptoms. These have also paved way for missions to discover alternative anti-osteoporotic agents that are comparable in effectiveness to estrogen but with minimal adverse effects. Several potential alternative agents were discovered including soy[
11], blueberry[
12],
Achyranthes bidentata[
13] and tocotrienol[
14]. Recently,
Labisia pumila var. alata (LPva), a herb used for women’s health, was found to produce beneficial effects similar to estrogen on bone biomarkers in postmenopausal osteoporosis animal model[
15]. These bone protective effects were further confirmed by bone histomorphometric analysis[
14‐
16] and bone biomechanical strength[
15‐
17].
Labisia Pumila (LP), a plant from the family of
Myrinaceae is a popular herb in Malaysia, Indonesia and Indo-China. There are three variants of
Labisia pumila, which are
var. alata (LPva),
var. pumila and
var. lanceolata[
18]. In Malaysia, it is known locally as “Kacip Fatimah” and the extract can simply be prepared by boiling the leaves, roots or the whole plant in water and the extract is taken orally[
18,
19]. Nowadays, various
Labisia pumila preparations are available commercially in the forms of capsules or added to drinks. It is used exclusively by women to shrink the uterus, facilitate labor, and improve menstrual irregularities and as post-partum medicine[
19,
20]. Its limited use by women for their health supplements has led to the belief that it is a phytoestrogen, a compound with similar chemical structure to estrogen[
21]. Several studies have demonstrated the estrogenic properties of LPva. It was found to inhibit estradiol binding to antibodies raised against estradiol[
22] and exert a specific estrogenic effect on human endometrial adenocarcinoma cells (Ishikawa-Var I line)[
23]. It mimicked estrogen action by preventing the shrinkage of the uterus due to estrogen deficiency in ovariectomized rats[
22] and initiate lipolysis in adipose tissue[
24]. LPva was also found to down-regulate 11β-hydroxysteroid dehydrogenase-1 expressions in liver and adipose tissues and also decrease serum corticosterone levels in ovariectomized rats[
25]. In terms of bone protection against estrogen deficiency, LP was reported to exert estrogen-like effects on bone remodeling[
15‐
17].
Bone remodeling involves a fine balance between bone formation and resorption. Any disturbance to the balance between these two processes would lead to bone pathology. There are several factors or cytokines known to play important roles in bone remodeling. Bone resorption is regulated by Receptor Activator of Nuclear Factor kappa-B ligand (RANKL) and Osteoprotegerin (OPG), which are produced by osteoblasts[
26]. RANKL binds to RANK receptors which are located on osteoclast precursors to promote differentiation into mature osteoclasts and activate their bone resorptive activity[
27]. It was reported that administration of serum RANKL to mice promoted osteoclast growth and activation, leading to osteoporosis[
28]. OPG acts as anti-resorptive decoy receptor by binding to RANKL and preventing it from binding to RANK receptors. As a result, OPG inhibits osteoclast differentiation and its bone resorptive activity. In a recent development, a fully human monoclonal antibody against RANKL was developed to inhibit osteoclast activity. This new agent named denosumab is still under clinical trial as a new anti-osteoporotic agent[
29].
Osteoclastogenesis also requires Macrophage-Colony Stimulating Factor (MCSF), which is also expressed by osteoblasts. It binds to the MCSF receptors situated in the osteoclasts and stimulates osteoclastogenesis, but the mechanism involved is still unclear[
30]. There are several factors which are known to affect osteoblast activity and differentiation. Among the important one is bone morphogenetic protein-2 (BMP-2), which promotes osteoblast differentiation and plays an important role in bone repair and regeneration[
31]. In summary, the differentiation and activation of osteoclast are influenced by the RANKL/OPG system and MCSF, while BMP-2 plays a key role in osteoblast differentiation. Estrogen was able to maintain bone density by regulating the gene expressions of these factors[
32] and LPva may have similar actions. To the best of our knowledge, there is no study on the mechanism of LPva extract in preventing bone loss due to estrogen deficiency. Therefore, the aim of the study is to determine the molecular mechanism of LPva in protecting bone against estrogen-deficient osteoporosis by measuring the bone-related gene expressions.
Discussion
Hormone replacement therapy (HRT/ERT) has been used for the treatment and prevention of postmenopausal osteoporosis, but it has been associated with serious side-effects (Ferguson, 2004)[
36]. Multiple studies have reported that women who took HRT have slightly higher rates of thromboembolism, heart attack, breast cancer, ovarian cancer, stroke, and Alzheimer’s disease[
8‐
10]. LPva has potential as an alternative to ERT for the treatment of postmenopausal osteoporosis. In terms of safety, several toxicity studies have confirmed that LPva is safe[
37,
38]. While, in terms of its action, LPva has demonstrated phytoestrogenic properties[
20,
21]. In an earlier study, LPva was found to reverse the bone biochemical marker changes due to ovariectomy[
15]. Following that, a further study reported that LPva protected bone from osteoporotic changes due to estrogen deficiency. This was based on its ability to preserve the bone histomophometric parameters of ovariectomised rats[
16]. These osteo-protective effects of LPva would be beneficial if they are accompanied by improvement in the bone strength, thus reducing the risk of fracture. This was confirmed by a biomechanical study which showed that supplementation of LPva in ovariectomised rats resulted in stronger femoral bone[
17].
Several mechanisms of the bone protective effects of LPva were proposed. Other than acting as phytoestrogen, LPva may exert anti-inflammatory and anti-oxidant effects[
17]. There were reports that inflammation may induce osteoporosis[
39,
40]. LPva may inhibit inflammation that may be responsible for osteoporosis by inhibiting tumor necrosis factor (TNF)-α production and down-regulating cyclooxygenase-2 expression[
41].
Reactive oxygen species were shown to cause bone loss by stimulating osteoclast differentiation[
42] and promoting osteoblast apoptosis[
43]. LPva exhibited anti-oxidative properties as it contains flavanoids, ascorbic acid, beta-carotene, anthocyanin and phenolic compounds[
44]. Beta-carotene was found to have the best correlation with anti-oxidative capacities of LP, followed by flavonoids, ascorbic acid, anthocyanin and phenolic content[
45]. Flavonoids were confirmed by phytochemical screening to be present in our LPva extract. It is a potent free radical scavenger in oxidative stress-related diseases such as osteoporosis and rheumatism[
46]. Other potent anti-oxidants such as vitamin E have also been shown to protect bone against osteoporosis[
14]. Therefore, the anti-oxidative and anti-inflammatory properties of LPva extract may have contributed to the effectiveness of this medicinal plant in treating osteoporosis.
In the final pathway for pathogenesis of osteoporosis, there will be an imbalance between bone formation by osteoblast and bone formation by osteoclasts with the latter getting the upper hand. RANKL/OPG system, MCSF and BMP-2 played an important role in the regulation of the osteoclastic and osteoblastic activities. Therefore, their gene expressions were measured in this study to better understand the mechanism of LPva. To the best of our knowledge, this is the first report on the molecular mechanism of LPv in preventing bone loss due to estrogen-deficient osteoporosis.
The function of OPG is to block the binding of RANKL to RANK receptors on committed pre-osteclastic cells[
47]. Therefore, OPG is a potent anti-osteoclastogenic factor. Estrogen is known to stimulate production of OPG, while, estrogen deficiency leads to down-regulation of OPG[
48]. As expected, in the present study, the OPG gene expression of the ovariectomised control group was found to be down-regulated. Both the LPva supplementation and ERT were able to revert back the OPG gene expression to sham levels. This study has shown that osteo-protective mechanism of LPva may be similar to ERT i.e. via stimulation of OPG production.
Estrogen deficiency led to up-regulation of pro-inflammatory cytokines such as TNF-α and interleukins[
49,
50]. TNF is an important cofactor of bone resorption as it supports osteoclasts activation mediated by RANKL and c-Fms/MCSF. RANKL is a membrane-bound molecule of TNF ligand family which promotes osteoclasts formation[
51]. In the present study, LPva may share similar mechanisms with ERT to protect bone as both were able to down-regulate the RANKL gene expression of ovariectomised rats.
This suggested a novel regulation of OPG and RANKL by LPva, which may help us to understand the mechanism of protection against estrogen-deficient bone loss. Interestingly, phytoestrogens such as genistein were also able to enhance osteoblastic OPG production through ER-dependent mechanisms and concurrently suppress RANKL gene expression which is associated with an inhibition of osteoclastogenesis[
52‐
55]. Therefore, the phytoestrogenic element in LPva could be the reason for these novel findings.
We did not find any significant change in the MCSF gene expression after ovariectomy. An
in vitro study of human endometrial stromal cells found that MCSF production was dose-dependently enhanced by the addition of sex hormone[
56]. However, in this study, both ERT and LPva did not produce any significant changes in the MCSF gene expression. This meant that M-CSF was not affected in this model of osteoporosis.
BMP-2 plays an important role in bone repair and regeneration[
31]. The BMP-2 gene expressions in the femora of ovariectomised rats were significantly increased by both LPva and ERT until they were similar to the sham level. Similar to our findings, Zhou
et. al[
57] also found that estrogen was able to activate BMP-2 gene transcription. Estrogen has been shown to stimulate the differentiation and activity of osteoblasts[
58,
59] and increase bone formation and bone mass in animal models[
60,
61]. Based on our results, the increased BMP-2 in LPva group was probably contributed by the phytoestrogenic effects of LPva.
Competing interests
The authors declare to have no conflict of interest whatsoever. The authors alone are responsible for the content and writing of this paper.
Authors’ contributions
ANS, INS and INM designed the study. SNF carried out the study and collected the samples. NM and NM participated in the statistical analysis. SNF and ANS drafted the manuscript. INS, INM, NM and NM read and edited the manuscript. All authors approved the final manuscript.