Benefits And Mechanisms Of Polysaccharides From Chinese Medicinal Herbs For Anti-osteoporosis Therapy

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Shan shan Lei a,1, Jie Su d,1, Yang Zhang a,1, Xiao wen Huang a, Xu ping Wang a, Min Cong Huang b,*, Bo Li c,*, Dan Shou a,d,**
a * Department of Medicine, Zhejiang Academy of Traditional Chinese Medicine, Hangzhou, Zhejiang 310007, PR China
b * Center of Safety Evaluation, Hangzhou Medical College, Hangzhou, Zhejiang 310053, PR China
c * Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China
d * Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, PR China

Abstract

Osteoporosis is a systemic metabolic bone disease with an increasing incidence rate. Chinese medicinal herbs have a long history of treating bone diseases. Polysaccharides are an important category of phytochemicals in Chinese medicinal herbs, and their health benefits have increased the interest of the public. Numerous studies have indicated that polysaccharides exhibit anti-osteoporosis effects by balancing bone resorption and bone formation, but the detailed effects and mechanisms have not been systematically summarized. We performed a comprehensive review of the literature to consolidate studies for the period 2000–2021 by conducting electronic searches on the PubMed, CNKI, VIP, and Wanfang databases. In total, polysaccharides from 19 kinds of Chinese medicinal herbs in 54 studies have shown bone homeostasis protective properties. In vivo and in vitro experiments have demonstrated that polysaccharides present properties in the treatment of postmenopausal osteoporosis, senile osteoporosis, and glucocorticoid-induced secondary osteoporosis, especially postmenopausal osteoporosis. Moreover, a number of signaling pathways, such as the Wnt/β-catenin signaling pathway, BMP/SMAD/RUNX2 signaling pathway, OPG/RANKL/RANK signaling pathway, apoptosis pathway, and transcription factors, are regulated by polysaccharides and participate in improving bone homeostasis. This review will provide a better understanding of the anti-osteoporotic effects of polysaccharides and the concomitant modulations of signaling pathways.

Keywords: Polysaccharides, Chinese medicinal herbs, Mechanisms, Bone homeostasis

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1. Introduction

Osteoporosis is a systemic metabolic bone disease characterized by microarchitectural deterioration, low bone mass, and an increased risk of fractures. According to the results of the first osteoporosis epidemiological survey conducted by the National Health Commission of China in 2018, the prevalence rate of osteoporosis in women over 50 years old reached 32.1%, while that in women over 65 years old was 51.6%. Approximately 10 million people over 50 years old in the United States have osteoporosis, and 34 million people are at risk of this disease. In the UK, 50% of women over 50 years old and 20% of men suffer from osteoporotic fractures [1]. Brittle fracture caused by osteoporosis can lead to increased morbidity and mortality, resulting in a huge social burden [2].

An imbalance in bone homeostasis is well known to be caused by more osteoclast-mediated bone resorption than osteoblast-mediated bone formation results in osteoporosis. Many factors lead to bone homeostasis, but the fundamental mechanism is an imbalance between osteoblasts and osteoclasts, including (i) decreased osteoblast (OB) differentiation and activity, resulting in reduced bone deposition; (ii) increased osteoclast (OC) differentiation and activity, resulting in excessive bone resorption; and (iii) inflammation and oxidative status [3]. In addition, the functions of OB and OC are regulated by a variety of transcription factors and signaling pathways.

In China, several Chinese medicinal herbs that possess functions to ease the joints and strengthen bones and muscles have been used for the treatment of osteoporosis for hundreds of years. Polysaccharides are one of the main active components of many Chinese medicinal herbs, such as Polygonatum sibiricum Delar. ex Redoute, Angelica sinensis (Oliv.) Diels and Morinda Officinalis How. Meanwhile, polysaccharides have been proven to have multiple functions, such as antioxidant [4], immunomodulation [5], and anti-fatigue functions [6]. To date, numerous studies have indicated that polysaccharides exhibit anti-osteoporosis effects by balancing bone resorption and bone formation, but the detailed effects and mechanisms have not been systematically summarized.

In this review, we collected a total of 54 studies of polysaccharides on anti-osteoporosis from 2000 to 2021 years, and polysaccharides from 19 kinds of Chinese medicinal herbs were researched. These studies included 25 in vivo experimental studies and 40 in vitro experiments. The experimental animal model included ovariectomized (OVX) rats, SAMP6 mice, and glucocorticoid-induced osteoporosis (GIOP) zebrafish. MCET3-E1 cells, BMSCs, dexamethasone-induced OB, and RANK-induced BMM cell models have been widely used in vitro experiments. The mechanisms of crude polysaccharides or purified polysaccharides on anti-osteoporosis were focused on OB or OC proliferation, differentiation, including expression of transcription factors, inflammatory factors, signals pathways such as Wnt/β-catenin signaling pathway, BMP/SMAD/RUNX2 signaling pathway, OPG/RANKL/RANK signaling pathway, etc. We reviewed the anti-osteoporosis effects and mechanisms of polysaccharides from 19 kinds of Chinese medicinal herbs and summarized these in Table 1.

2. Polysaccharides derived from Chinese medicinal herbs for anti-osteoporosis

Among the 19 kinds of herbs, 13/19 were from the rhizome part, 1/19 from the flower, 2/19 from a leaf part, 1/19 from a fruit part, and 2/19 from fungus. All of the evidence of polysaccharides from these Chinese medicinal herbs to improve bone homeostasis and limit bone loss is reported in the following paragraphs.

2.1. Polysaccharides derived from rhizomes of Chinese medicinal herbs

Achyranthes bidentata Blume (AC, “Niu xi”), Morinda Officinalis How (MO, “Ba-Ji-Tian”), Curculigo orchioides Gaertn (CO, “Xian Mao”), Cynomorium solarium Rupr. (CY, “Suo yang”), Cistanche deserticola Ma (CD, “Rou Cong Rong”), and Cibotium barometz (L.) J. Sm (CB, “Gou Ji”) are Chinese medicinal herb that “tone the kidney and strengthen bone” and are especially applied for the treatment of postmenopausal osteoporosis. The main active compounds of polysaccharides are reported to have exact beneficial effects for anti-osteoporosis in OVX rat models. Crude polysaccharides from AC [7,8], MO [9–11], CO [12], CY [17], and ABPB-3 [13] (a purified polysaccharide from AC) have been reported to increase bone mineral density (BMD), improve biomechanical indices of femurs in OVX rats, and regulate the microarchitecture of trabecular bone after continuous administration for almost 3 months. Meanwhile, ABPB-3 could also increase serum osteocalcin (OCN) [13], bone glutamate protein (BGP), and urine deoxypyridinol (DPD) levels [15]. In an in vitro experiment, polysaccharides from CB (CBP) and a novel homogeneous heteropolysaccharide COP70–3 from CO increased alkaline phosphatase (ALP) activity and enhanced the mineralization rate [12] [16]. Another purified polysaccharide, COP90–1, from CO promoted the proliferation and differentiation of primary mouse OB [17]. Furthermore, two novel polysaccharides of ABW50-1 and ABPB-3 from AC notably increased bone mass in GIOP zebrafish models [18] [13]. Polysaccharides from CD (CDP) remarkably ameliorated bone histopathological damage and promoted the formation of new bone in SAMP6 mice [45].

Astragali Radix (AS, “Huang qi”), Angelica sinensis (Oliv.) Diels (AN, “Dang Gui”), and Hedysarum Polyporus Hand.-Mazz. (HP, “Hong qi”) are Chinese medicinal herbs that “supplement qi and nourish blood”. AS polysaccharides (ASP) have been reported to be able to reverse the decrease in bone mass, increase serum ALP and BGP, and regulate biomechanical indices of the femur in OVX mice and Dex-induced and stress-induced osteoporosis rats in a dose-dependent manner [20,21]. Polysaccharides from AN (ANP) increased the number of trabeculae and promoted the repair of bone injury in type 2 diabetic rats. In vivo study, 40 mg/L ASP increased the calcium nodule number and ALP activity in BMSCs induced by 25.5 mmol/L glucose [22]. ANP and polysaccharides from HP (HPP) appear to improve the proliferation of OB and enhance ALP activity in a dose-dependent manner [23,24].

Polygonatum sibiricum Delar. ex Redoute (RP, “Huang Jing”) and Dendrobium officinale Kimura et Migo (DO, “Tie pi shi hu”) are Chinese medicinal herbs that “nourish Yin”. In vivo studies have demonstrated that polysaccharides from RP (RPP) could significantly increase the whole BMD and improve the bone trabecular microstructure of OVX rats after continuous administration [25,26] and could also improve symptoms in an osteoporotic fracture model [27]. DO polysaccharides (DOP) prevented the degradation of trabecular microstructural and improved BS/TV, Tb. N, and the reduction in Tb.Sp age-induced osteoporosis model [28]. Likewise, daily administration of polysaccharides from Gastrodia elata Bl. (GA, “Tian ma”) and Saposhnikovia divaricata (Turcz.) Schischk. (SA, “Fang Feng”) also increased BMD [29,30] and serum Ca2+, Mg2+, and P2− levels [31] in the OVX model.

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2.2. Polysaccharides derived from the leaves of Chinese medicinal herbs

Epimedium brevicornum Maxim (EB, “Yin Yang Huo”) is a very popular leaf, traditionally used to treat bone diseases and gonad dysfunction in Chinese medicine for thousands of years. EB could relieve post-menopausal symptoms and inhibit osteoporosis and other bone loss diseases. A recent in vitro study indicated that EB polysaccharide (EBP) could stimulate and improve the proliferation and differentiation of OB cells induced by dexamethasone [32].

Hairyvein Agrimonia Herb and Bud (AP, “Xian he cao”) belongs to Rosaceae and is used as Chinese medicine for the treatment of diabetes and arrhythmia. A water-soluble polysaccharide (APP-AW) was isolated from AP, and three sulfated derivatives (S1, S2, and S3) from APP-AW could inhibit apoptosis in a dexamethasone-induced OB model [33].

2.3. Polysaccharides derived from fungi and mushrooms of Chinese medicinal herbs

The Chinese medicine of Poria cocos (Schw.) Wolf. (PO, “Fu ling”) has famously been used for daily diet and medicine in China and other Asian countries from ancient years. As a classical anti-oxidant agent, PO more recently exhibited benefits for the treatment of angina pectoris, hypertension, and bone metabolism. PO polysaccharide (POP) is an active and primary compound extracted from PO. An in vivo study indicated that POP treatment impaired RANKL-induced OC formation in RAW264.7 and BMMS cells in a dose-dependent manner and attenuated resorption activity [34].

Polystictus versicolor (L.) Fr. (TV, “Yun Zhi”) is one of the most popular medicinal mushrooms from Coriolus Versicolor(L. ex Fr.) Quel due to its various biologically active components. Polysaccharopeptides from TV (TVP) have been shown to have immune-regulatory functions in normal and type I diabetes mellitus (DM) rats. A recent study demonstrated that TVP was an effective treatment for osteoporosis in diabetes mellitus rats. Compared with the model group, TVP mitigated DM-induced bone deterioration, as determined by increasing the bone volume of the proximal tibia, trabecular number, and femoral bone strength (11% maximal load, 22% stiffness, and 14% modulus) and by reducing the femoral cortical porosity by 25% [35].

2.4. Polysaccharides derived from other Chinese medicinal herbs

Lycium chinense Miller (LY, “Gou qi”), belonging to Lycium barbarum L., is widely distributed in northwestern China, and its fruits are recorded to play a role in intervening in postmenopausal metabolism. Rodent models of postmenopausal estrogen deficiency-induced osteoporosis and dexamethasone-induced osteoporosis have shown that LYP could provide effective treatment for osteoporosis. In vivo studies have demonstrated that LY polysaccharide (LYP) significantly increased

lumbar BMD and the dry weight of femurs in OVX rats after treatment for 12 weeks, improved trabecular bone structure, and reduced lacunal absorption [24]. Moreover, LYP also increased blood calcium, ALP content, and BMD in glucocorticoid-induced osteoporosis rats [36].

Carthamus Flos (SF, “Hong Hua”) from Carthamus tinctorius L. has been used as Chinese medicine for the treatment of stroke and coronary heart disease. Modern pharmacological experiments have also demonstrated that polysaccharides isolated from SF (SFP) possess potential therapeutic value for the treatment of osteogenesis imperfect and osteoporosis. In vitro studies showed that SFP could dose-dependently enhance the proliferation of primary OB and mineralization and that this enhancement was accompanied by increased ALP activity and collagen synthesis expression [37].

3. Mechanism by which polysaccharides improve bone homeostasis by regulating OB and OC

3.1. Mechanism by which polysaccharides promote osteoblast-mediated bone formation

OB undergo four stages in bone formation, including osteoblast proliferation, extracellular matrix maturation, extracellular matrix mineralization, and osteoblast apoptosis. Many factors, such as signaling pathways and transcription factors can regulate these stages and ultimately regulate bone formation. Here, we summarize the transcription factors and signaling pathways by which polysaccharides affect bone formation in OB. The mechanisms of OB-mediated bone formation and polysaccharide regulation of OB function that are involved in our research are shown in Figs. 1 and 2.

Fig. 1. Summary mechanisms of osteoporosis that are involved in our research. Promote (→), inhibit (⊥). In the BMSCs, the binding of Wnt proteins with membrane-bound frizzles receptors and LRP5/6 co-receptor initiates Axin1 complex inhibit the ubiquitination and degradation of β-catenin and accelerate its translocation into the nucleus [38]. This nuclear β-catenin binds to and co-activates members of the T-cell factor/lymphoid-enhancing factor family of transcription factors (TCF/LEF) activate downstream target genes, such as Runx2 and Osterix (Osx), thus leading to a promotion for differentiation and proliferation of OBs [32]. DKK-1 inhibits Wnt bind to frizzled receptors and LRP5/6, the wnt/β-catenin signaling pathway is blocked. In the OB, bone morphogenetic proteins (BMP) bind to the BMP receptor (BMPR), activate SMAD1/5/8 proteins that enter into the nucleus, then regulate RUNX2 gene expression. Runx2 can combine with core-binding factor β to form a heterologous dimer structure and then bind to DNA, inducing the expression of bone formation-related genes, such as Osx, Bone glutamate protein (BGP), and ALP. PIP3 produced by PI3K activation binds to the PH domain at the N-end of Akt, translocates Akt to the plasma membrane, which catalyzes Akt ser473 and Thr308 sites phosphorylation. After Akt activation, it promotes the expression of downstream pro-apoptotic factors of bax-2, caspase 3 and inhibits the anti-apoptotic factor of BCL-2. In the pre-OC, IL-1, IL-6, and TNF-α active NF-κB, which improves the translocation of NF-κB into the nucleus, which induces differentiation from pre-OC to OC. In the OC, IL-6 binds to interleukin 6 receptor (IL-6R), TNF-α binds to TNF-α receptor (TNFR), RANKL binds to RANK and further recruits TNFR-related factors (TRAFs) to initiate downstream signaling cascades, including NF-κB phosphorylation, p38 phosphorylation, JNK, and ERK1/ 2. The final result of RANKL-RANK signaling is the activation of osteoclast transcription factors, such as NF-κB, activator protein 1 (AP-1), Proto-oncogene protein (FOS), and activated nuclear factor of activated T-cells (NFATc1). NFATc1 induced the expression of osteoclast-specific genes such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K (Ctsk), which leads to differentiation and promotes OC maturation.

Fig. 2. Summary of polysaccharides and possible mechanisms that are involved in the treatment of osteoporosis. ACP: polysaccharide from Achyranthes bidentata Blume; ASP: polysaccharide from Astragali Radix; ANP: polysaccharide from Angelica Sinensis (Oliv.) Diels; APP: polysaccharide from Hairyvein Agrimonia Herb and Bud; COP: polysaccharide from Curculigo orchioides Gaertn; CYP: polysaccharide from Cynomorium songaricum Rupr; CDP: polysaccharide from Cistanche deserticola Ma; CBP: polysaccharide from Cibotium barometz (L.) J. Sm; DOP: polysaccharide from Dendrobium officinale Kimura et Migo; EBP: polysaccharide from Epimedium brevicornum Maxim; WSS25: polysaccharide from Gastrodia elata Bl.; HPP: polysaccharide from Hedysarum Polyporus Hand.-Mazz.; LYP: polysaccharide from Lycium chinense Miller; MOP: polysaccharide from Morinda Officinalis How; POP: polysaccharide from Poria cocos (Schw.) Wolf.; RPP: polysaccharide from Polygonatum sibiricum Delar. ex Redoute; SAP: polysaccharide from Saposhnikovia divaricata (Turcz.) Schischk.; SFP: polysaccharide from Carthami Flos.; TVP: polysaccharide from Polystictus Versicolor (L.) Fr.

3.1.1. Transcription factors

Osteogenic differentiation of BMSCs is regulated by various transcription factors, such as Runt-related transcription Factor 2/core binding factor α1 (Runx2/Cbfa1) and osterix. Cbfα1, also known as polyomavirus enhancer-binding protein 2 α A (PEBP2 αA) or acute myeloid leukemia Factor 3 (AML3), is a transcription factor of the runt structural gene family. Osterix, a critical factor that controls downstream targets in bone formation, is an important regulator of OB differentiation and is the earliest and most specific marker of OB differentiation.

MOP-mediated serum intervention of BMSCs for 14 days upregulated the expression of Cbfα1 mRNA and promoted osteogenic differentiation [39]. Another study showed that the expression of Cbfα1 mRNA in rat calvarial OB was up-regulated after treatment with a water extract of Morinda Officinalis and MOP-containing serum for 72 h, and the effect of MOP was better than the effect of a water extract of Morinda Officinalis [40]. MOP administered to OVX osteoporosis rats for 30 days increased the expression of the tibial genes Cbfα1 and DMP1 [10]. A new inulin fructan MOW90-1 isolated from MO90 up-regulated the expression of Runx2 and Osterix and promoted the proliferation, differentiation, and mineralization of MC3T3-E1 cells [14].

A recent in vitro study assessed the effect of HPP on the osteogenic differentiation of MC3T3-E1 osteoblastic cells and found that HPP upregulated ALP activity in a dose-dependent manner and increased the expression and transcriptional activity of Runx2 and Osterix, the two master genes of OB differentiation [24]. Liao et al found that ANP could notably up-regulate the mRNA expression of CyclinD1, Runx2, and Osterix [22,23]. ASP also increased the expression of CyclinD1 and promoted proliferation [41].

3.1.2. Wnt/β-catenin signaling pathway

The Wnt/β-catenin signaling pathway plays an important role in the self-renewal, directional differentiation, preosteoblast proliferation, OB formation, and apoptosis of BMSCs. In the process of promoting OB differentiation, Wnt controls OB differentiation genes by regulating the level of β-catenin in BMSCs. Activated Wnt signaling could promote β-catenin aggregation, resulting in OB differentiation and bone formation. The expression of Wnt3a and Wnt10b in BMSCs can activate the Wnt/β-catenin signaling pathway and promote the proliferation of BMSCs [42]. DKK-1 is an inhibitor of the Wnt signaling pathway, inhibits bone formation, and leads to osteoporosis while antagonizing DKK-1 can increase bone mass [43].

MOP increased the proliferation rate of OBs in vitro, increased ALP activity, and down-regulated the protein expression of DKK-1 [44]. Further in vivo investigation of the molecular mechanisms behind the anti-osteoporosis effect of CDP indicates that the attenuation of bone degeneration is associated with activating the Wnt/β-catenin signaling pathway [45]. RPP intervention improved the ALP activity of BMSCs in a dose-dependent manner, significantly enhanced the mineralization ability of cells, significantly increased the expression of ALP, Runx2, and Osteocalcin genes in BMSCs, significantly up-regulated the expression of β-catenin, and promoted the high expression of TCF binding sites. The mechanism may be related to activating the Wnt/β-catenin signaling pathway and promoting the differentiation of BMSCs into OBs [46], but RPP had no effect on the BMP pathway. Findings also demonstrated that RPP promoted OB differentiation and mineralization by regulating the ERK/GSK-3β/β-catenin signaling pathway [47]. Li et al. found that BMP9 is the direct target of miR-152, and ASP could down-regulate miR- 152 expressions and promote BMP9 up-regulation, thereby activating PI3K/AKT and Wnt/β-catenin pathway signaling in BMSCs [41]. ASP alleviated oxidative stress-induced osteoporosis by regulating the FoxO3a/Wnt2/β-catenin signaling pathway [48]. LYP-mediated serum activates the expression of Wnt signaling pathway-related proteins β-catenin and Wnt10b promotes the differentiation of BMSCs into OB, and increases mineralized nodules [49].

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3.1.3. BMP/SMAD/RUNX2 signaling pathway

The BMP/SMAD signaling pathway plays an important role in activating bone formation gene transcription. Bone morphogenetic protein II (BMP2), a member of the TGF-β superfamily, is highly expressed in OB and regulates differentiation both in vitro and in vivo [50]. BMP binds heterodimeric receptors to activate SMAD proteins, which transactivate osteoclastogenic genes either directly or via Runx2 [51]. Runx2 is the downstream target gene of the BMP2/Smad signal transduction mechanism and a transcription factor required for osteoblastogenesis. Runx2 can combine with core-binding factor β to form a heterologous dimer structure and then bind to DNA, inducing the expression of bone formation-related genes, such as COL-І, BGP, and ALP.

Huang et al isolated two novel polysaccharides, CBP70-1-1 and CBP70-1-2, from rhizomes of Cibotium barometz and found that they could promote OB proliferation at a low concentration. CBP70-1-1 at 3.93 and 7.86 μM and CBP70–1-2 at 1.73 and 6.92 μM significantly increased ALP activity and mineralized nodules in MC3T3-E1 cells. Moreover, CBP70-1-2 increased the expression and transcriptional activity of BMP2, and the increased expression of BMP2 subsequently stimulated the downstream gene Smad1. The phosphorylation of Smad1 is increased, which further stimulates the osteogenic marker gene Runx2 to promote bone formation [50].

Another study demonstrated that CEP could improve bone information by upregulating the protein expression of BMP2/Smad1 and Smad5/Runx2 [52]. Chen et al found that Gastrodia elata Bl. polysaccharide WSS25 could inhibit the expression of BMP2/Smad1 pathways and inhibit osteoclastic differentiation [29]. RPP promoted bone formation in OVX rats by increasing the levels of ALP, OPG, and BGP and the protein expression of BMP2 [26]. APP enhanced ALP activity, promoted proliferation and differentiation of OB, and increased OB differentiation marker proteins BMP2, Runx2, Osterix, and Osteocalcin. The molecular mechanism was related to the enhanced miR-70 expression [53] and regulation of the Wnt/β-catenin signaling pathway [54].

3.1.4. Antiapoptosis mediated pathways

Apoptosis, a form of programmed cell death, plays a central role in tissue homeostasis, and deregulation of apoptosis has been implicated in numerous pathological conditions [55]. Proteins of the Bcl-2 family have either pro- or antiapoptotic activities and regulate the mitochondrial pathway of apoptosis by controlling mitochondrial outer membrane permeabilization (MOMP). MOMP is the pivotal event in the intrinsic apoptotic pathway. Bax, which participates in forming MOMPs, leads to the release of the contents of the mitochondrial intermembrane space into the cytoplasm. The released cytochrome c binds to a cytosolic protein containing a caspase-recruitment domain (CARD) to form the apoptosome and subsequently to recruit multiple procaspase-9 molecules, finally activating downstream executioner caspases such as caspase-3. Anti-apoptotic proteins of Bcl-2 block cell death by preventing the activation and homo-oligomerization of Bax [56]. Moreover, Bax protein is regulated by phosphorylation in an Akt-dependent manner, and this phosphorylation event inhibits the effects of Bax on mitochondria by maintaining it in the cytoplasm [57].

Glucocorticoids are widely used to treat inflammatory diseases and result in approximately 30% OB cell apoptosis in patients with osteoporosis caused by excessive application of glucocorticoids. A water-soluble polysaccharide (APP-AW) was isolated from Agrimonia pilosa, and three sulfated derivatives (S1, S2, and S3) from APP-AW inhibited apoptosis in a dexamethasone-induced OB model [33], reversed the increase in Bax, cytochrome, and caspase-3 and decreased Bcl-2 and c-Myc protein expression in MC3T3-E1 cells. An in vitro study showed that MOP inhibited OB apoptosis induced by all-trans retinoic acid [58]. SFP appears to protect OB cells from dexamethasone-induced apoptosis, possibly due to its effect on reversing the activation of caspase-3 and cleavage of PARP [37]. EBP has up-regulated the expression of Bcl-xl and Bcl-2 and decreased Bax and caspase-3 expression via the PI3K/ Akt/mTOR signaling pathway in dexamethasone-induced OB [32].

3.1.5. Oxidative stress-mediated pathways

Enhancement of the oxidative stress response during aging may be an important factor causing osteoporosis. Oxidative stress is a pathological condition that implies overproduction of reactive oxygen species (ROS) under conditions when their elimination is reduced. As an indicator and regulator of oxidative damage, the nuclear factor erythroid 2- related factor 2 (Nrf2)/Heme Oxygenase 1 (HO-1) pathway, which protects against oxidative damage and cell death, has been the primary focus of research for many years. An in vivo study showed that DOP could rescue the H2O2- induced switch of BMSC differentiation fate and increase the expression of Nrf2, HO-1, and Nqo1 [28].

3.1.6. Promotes bone-specific matrix and bone mineralization

Bone sialoprotein (BSP), osteopontin, and osteocalcin are phenotypic markers for mature OB and important markers of matrix mineralization. Osteopontin is secreted by OB and participates in the mineralization of the bone matrix, which can combine with hydroxyapatite through a region rich in aspartic acid and participates in the mineralization of the bone matrix. BSP, a major extracellular noncollagenous matrix protein in bone tissue localized to the mineralized matrix, can promote the nucleation of hydroxyapatite mineralization in vitro and increase calcium incorporation and nodule formation. Osteocalcin, produced and secreted almost exclusively by OB, binds to Ca2+ and is responsible for the mineralization of the bone matrix [59].

MOP (50 μg⋅mL-1) promoted MC3T3-E1 cell proliferation, promoted the secretion of collagen type 1 (COL-І) and Osteocalcin, and enhanced ALP activity [60]. RPP improved the biomechanical properties and BMD of osteoporosis fracture in rats by reducing the content of bone ALP, TRAPa, and TNF-α in osteoporosis of rats [25] and increasing the levels of ALP, OPG, and BGP and the protein expression of BMP2 [26]. A purified polysaccharide ABW70-1, isolated from radix achyranthis bidentatae, promoted the proliferation of MC3T3-E1 cells, increased ALP activity, mineral nodule formation, and gene expression of Osterix, Osteocalcin, and BSP, and promoted osteogenic differentiation [8].

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3.2. Mechanism by which polysaccharides inhibit osteoclast-mediated bone resorption

Bone resorption is the result of the interaction between activated OC and the bone matrix. The role of OC in bone resorption includes mainly four stages: osteoclast adhesion and polarization, bone resorption microenvironment acidification, bone matrix degradation, and endocytosis transport. In the process of osteoclast adhesion and polarization, osteoclast precursors are recruited to the surface of bone under the action of β-integrin and differentiate into OC stimulated by M-CSF, RANKL, and cytokines. In this process, transcription factors and cytokines such as IL-1 and IL-6 can also promote differentiation. Subsequently, OC generates an acidic microenvironment between the cell and the surface of the bone [61] and mature OCs secrete a variety of enzymes, including protein kinase K, matrix metalloproteinases (MMPs), and tartrate-resistant acid phosphatase [62] to degrade the bone matrix. The mechanisms of osteoclast-mediated bone resorption and polysaccharide regulation of OC function that are involved in our research are shown in Figs. 1 and 2.

3.2.1. Transcription factors

The differentiation of OC is regulated by various transcription factors. During osteoclastogenesis, NFATc1, which belongs to the NFAT transcription superfamily, is generally accepted to be an essential transcription factor that regulates RANKL-induced osteoclastogenic gene expression [63]. NFATc1 is an upstream nuclear transcription factor that plays a key role in regulating the expression of many osteoclast-specific genes [64], such as tartrate-resistant acid phosphatase (TRAP), β3- integrin, cathepsin K (Ctsk), and MMP-9, which are involved in the regulation of osteoclast differentiation, fusion, and activation. Meanwhile, Ctsk and MMP-9 degrade the organic bone matrix and contribute to bone resorptive activity [65].

An in vivo study demonstrated that WSS25 (GAP) could inhibit osteoclastic differentiation in the early stage and inhibit the expression of the osteoclast formation-specific genes TRAP, NFATc1, MMP-9, and CtsK in RANKL-induced RAW264.7 cells in a dose-dependent manner [29]. Further research found that the molecular mechanism of WSS25 in osteoclast formation was through inhibition of the expression of the BMP2/Smad1 pathway [29]. RPP down-regulated osteoclastic activity in a dose-dependent manner, including decreased expression of TRAP, MMP-9, CtsK, and NFATc1 in RANKL-induced RAW264.7 cells [66]. In addition, RPP down-regulated miR-1224 expression and the key targeting molecule Limd1 in the Hippo signaling pathway in bone marrow-derived macrophages (BMMs) [67]. Additionally, an in vitro investigation showed that CDP inhibited RANKL-induced osteoclast differentiation from BMMs and suppressed osteoclast fusion by inhibiting hydroxyapatite resorption activity and downregulating osteoclast marker mRNA levels of NFATC1, MMP9, Ctsk, and TRAP [19]. Another study reported that RPP also down-regulated the expression of specific genes, including TRAP, MMP-9, CtsK, and NFATc1, during OC formation [66].

3.3.2. OPG/RANKL/RANK signaling pathway

The OPG/RANKL/RANK axis system is a key factor in regulating osteoclast differentiation and bone resorption [68]. Rank, also known as TNFR superfamily member 11a, plays a crucial role in osteoclastogenesis. RANKL binds to rank and further recruits TNFR-related factors to initiate downstream signaling cascades, including p38, JNK, and ERK. The final result of RANKL-RANK signaling is the activation of osteoclast transcription factors such as NF-κB, activator protein 1 (AP- 1), cyclic adenosine monophosphate response element-binding protein (CREB), and activated t-nuclear factor-1 (NFATc1), all of which induce the expression of osteoclast markers, such as TRAP, β3 integrin, and CtsK [69]. OPG is a member of the TNF receptor superfamily and is also known as osteoclastogenesis inhibitory factor (OCIF), which can competitively antagonize RANKL, thus inhibiting the differentiation of OC [70].

ACP dose-dependently increased bone formation markers of serum OC and BAP and decreased bone resorption markers of serum TPACP5b, NTX, and CTX in OVX rats, and the mechanism may be related to the increase of OPG and RANK protein expression in bone marrow tissue and the decrease of RANKL expression, thus resulting in inhibition of bone resorption [7]. Moreover, ACP inhibited RANKL-induced phosphorylation of the MAPK pathway [71]. CYP [15] increased the serum level of OPG and decreased RANKL and the ratio of RANKL/OPG in OVX osteoporosis model rats. ASP increased the serum level of OPG, decreased RANKL and the ratio of RANKL/OPG, and significantly decreased the levels of TNF-α and IL-2, resulting in the inhibition of osteoclast differentiation activity [20,21].

3.3.3. ERK/JNK signaling pathway

The ERK/JNK signaling pathway is involved in cell proliferation and differentiation, and phosphorylation-activated ERK1/2 translocates from the cytoplasm to the nucleus, which mediates the transcriptional activation of AP-1, c-Fos, and c-Jun and promotes the proliferation and differentiation of OC. Down-regulation of p38, ERK, and JNK inhibited the expression of NFAcT1 and c-Fos in RANKL-induced osteoclastogenesis. Treatment with ERK or JNK inhibitors significantly decreased the number of OC and IL-6 production [72]. Potential mechanisms of POP in osteoclastogenesis were matched to JNK and ERK signaling pathways (suppressed ERK1/2 phosphorylation, STAT3, and JNK1/2 expression) [34]. To confirm the effective treatment for osteoporosis, further in vivo research is necessary.

3.3.4. Regulated inflammatory factors

Many inflammatory factors promote OC differentiation by mediating RANKL expression [73]. TNF-α is a potent inducer of bone resorption and plays an important role in bone metabolism, which can directly induce the formation of OC from precursors OC in the presence of M-CSF or the absence of RANKL by activating NF-κB signaling [74] and induce RANK expression in OC precursors [75]. The pro-inflammatory cytokine IL-1β is a powerful stimulator of OC differentiation and bone resorption by inducing RANKL expression [76]. IL-6 induces osteoclastogenesis and OC activation via induction of RANKL expression in OB and stromal cells [77].

Zhu et al. found that MOP reduced serum IL-6 and TNF-α expression levels [10]. Ren et al. indicated that LYP administration for 90 days significantly reduced the IL-6 content in serum [78]. ASP has a protective effect on bone loss in OVX mice by significantly reducing the concentrations of TNF-α and inhibiting osteoclastogenesis [20]. Following treatment with ASP, the enhanced levels of TNF-α and IL-2 were significantly decreased in a dexamethasone-induced osteoporosis rat model, and the anti-inflammatory effect was related to the regulation of intestinal flora [21]. SA Polysaccharide (SAP) administration for 70 days significantly reduced the serum levels of IL-6 and TNF-α [30,31]. Others reported that RPP could inhibit bone resorption by reducing the content of TRAPa and serum IL-1, IL-6, and TNF-α in osteoporotic rats [25,27].

Cistanche tubulosa anti-osteoporosis

4. Conclusions

In conclusion, in vivo, and in vitro experiments have demonstrated that polysaccharides present properties for the treatment of postmenopausal osteoporosis, senile osteoporosis, and glucocorticoid-induced secondary osteoporosis, especially postmenopausal osteoporosis, by promoting OB differentiation and activity and decreasing OC differentiation and activity. The mechanisms by which polysaccharides improve bone homeostasis include mainly the Wnt/β-catenin signaling pathway, BMP/SMAD/RUNX2 signaling pathway, OPG/RANKL/RANK signaling pathway, ERK/JNK signaling pathway, apoptosis pathway, and transcription factors. This review will provide a better understanding of the anti-osteoporotic effects of polysaccharides and the modulation of signaling pathways, which is useful for providing a theoretical basis of polysaccharides for clinical application.

5. Future prospects

Several studies had unequivocally demonstrated the beneficial effects of polysaccharides on osteoporosis in vitro and in vivo experiments. At present, although there are no clinical researches reported on the treatment of osteoporosis with polysaccharides from Chinese medicinal herbs, a lot of clinical applications of polysaccharides have been explored and evaluated in the treatment of other diseases, such as tumor [79], Type 2 Diabetes [80]. Among them, Poria cocos polysaccharide, Astragali Radix polysaccharide, and Lycium chinense Miller polysaccharide have been approved by CFDA as raw materials. The Chinese medicinal herbs summarized in this paper have a long history in the clinical treatment of bone diseases, in vitro and in vivo experiments also show that the polysaccharides extracted from these herbs have an effect on osteoporosis patients. Therefore, we consider that further clinical research to confirm their anti-osteoporosis effect will help to promote the development of Chinese medicine herbs polysaccharides into functional foods or drugs.

However, in the process of the development of polysaccharides from Chinese medicinal herbs, there are several points that need to be considered and studied further. Firstly, polysaccharides possess the characteristics of a large molecular weight and complex structure, further study to explore the relationship between the biological activities of polysaccharides and chemical structures is needed.

In addition, more attention should be given to the effect of polysaccharides on gastrointestinal tract intestinal flora. Polysaccharides are represented by their low bioavailability, which depends upon degradation occurring at the level of first-pass metabolism and absorption from the gastrointestinal tract [81]. Numerous studies have found that the gastrointestinal tract is involved in the occurrence and development of osteoporosis [82–84]. Whether and how polysaccharides participate in bone metabolism by regulating intestinal flora is still unclear and may present a new polysaccharide strategy for the treatment of osteoporosis.

Acknowledgments

The research work was financially supported by the National Science Foundation of China (No. 82003977, 81803760, 82004022), the Zhejiang Province Excellent Young Talents Fund Project of Traditional Chinese Medicine (No. 2020ZQ011), and the Major Projects of Traditional Chinese Medicine in Zhejiang Province (2018ZY003), Basic Public Welfare Research Program of Zhejiang Province (LGF20H060006), Medical and Health Science and Technology Program of Zhejiang Province (2020KY507).

Chinese Medicinal herb--Cistanche, is good for anti-osteoporosis

From:' Benefits and mechanisms of polysaccharides from Chinese medicinal herbs for anti-osteoporosis therapy' by Shan shan Lei a, et al.

---International Journal of Biological Macromolecules 193 (2021) 1996–2005