Tonifying Shen-Yin and -Yang Principles in Treating Osteoporosis: All Roads Lead to Rome

Home , Shen (Chinese religion)

DOI: 10.15806/j.issn.2311-8571.2016.0031 World J Tradit Chin Med 2016; 2(4): 38–48 Research on TCM Theory Tonifying Shen-Yin and -Yang Principles in Treating Osteoporosis: All Roads Lead to Rome

Wei Goua,b, Nan-Mei Liua,b, Cheng-Long Wangc, Hui-Ling Wanga,b, Shi Lua,b, Dong-Feng Zhaoc* and Xiu-Zhi Yua,b* aDepartment of Dialysis, the 455th Hospital of People’s Liberation Army of China, Shanghai, 200052, China bDepartment of Nephrology, the 455th Hospital of People’s Liberation Army of China, Shanghai, 200052, China cLonghua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, China *Correspondence: Xiuzhi Yu, MD, PhD, Room 306, Inpatient Department, Department of Dialysis, The 455th Hospital of People’s Liberation Army of China, 338 West Huaihai Road, Shanghai, 200052, China. Tel: 86-21-81815134, Fax: 86-21-81815010, E-mail: [email protected]; Dongfeng Zhao, MD, PhD, Room 809, NO.12 Building, Spine Institute, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, 725 South Wanping Road, Shanghai, 200032, China. Tel.: 86-21-33324000, Fax: 86-21-64434704, E-mail: [email protected]

ABSTRACT

Tonifying Shen strategy works as an important alternative and complementary method that is widely used to treat osteoporosis in Traditional Chinese Medicine (TCM) based on the proper identification of Zheng. Shen deficiency Zheng is one of the main types of osteoporosis while Shen-Yin and -Yang deficiencies represent two basic principles/types of Shen deficiency. Currently, Tonifying Shen strategy, in particular, the Tonifying Shen-Yin and -Yang principles, has been demonstrated to exert osteoprotective effects. However, the mechanisms by which Tonifying Shen strategy and/or Tonifying Shen-Yin and -Yang principles function in treating osteoporosis are still not clearly understood. Here we first briefly explore current understanding of Tonifying Shen strategy, such as Tonifying Shen prescriptions, herbs, and effective components, in treating osteoporosis. Furthermore, the mechanism of Tonifying Shen-Yang principle in treating osteoporosis is reviewed from the clinic experience, animal, and mechanistic investigation. Moreover, the mechanisms by which Tonifying Shen-Yin principle in treating osteoporosis are established. Finally, we compare the similarity and difference between Tonifying Shen-Yang and -Yin principles in treating osteoporosis. Our findings indicate that both principles exert bone-protective effects in treating osteoporosis by simultaneously stimulating the differentiation of mesenchymal stem cells (MSCs) into osteoblast and the differentiation of hematopoietic stem cells (HSCs) into osteoclast. However, Tonifying Shen-Yin principle is in favor of increasing bone mineral density (BMD) while Tonifying Shen-Yang principle is in favor of osteoporosis-related syndromes. Our findings indicate that Tonifying Shen-Yin and -Yang principles not only share some similarity but also obtain some difference in treating osteoporosis. Key words: Osteoporosis, Shen-Yin and -Yang Deficiency, Tonifying Shen-Yin and -Yang, Osteoclast, Osteoblast Abbreviations: 1.25(OH)D3:1,25-dihydroxyVitaminD3;ALP:Alkalinephosphates;Beta2-MG:Beta2-microglobulin;BGP:Boneglaprotein; BGSSC: Bugushengsui capsule; BMD: Bone mineral density; BMP: Bone morphogetic protein; BSA: Bone sialoprotein; BSF: Bushenfang; BSJG: Bushenjiangufang; BSSF: Bushenshenggufang; BSYG: Bushengyanggupian; BGSSC: Bugushengsuipian; Col-1: Collagen type I; CORT: Corticosterone; DM:Dexamethasone;DRG:DriedrootofRehmanniaglutinosa;FLL:Fructus Ligustri Lucidi, one effective ingredient form herb; GLP: Gulin Pill; HSC: Hemateopoeitic stem cell; IGF-1: Insulin-likefactor-1; IKSBC: Bushenjiangujiaonang; IL-1,6,7:Interleukin(IL)-1,6,7;LPS:Lipopolysaccharide; LWDHP: Liuweidihuangpills; MAPK: Mitogen-activated protein kinase; MSC: Mesenchymal stem cell; M-CSF: Macrophage Colony-Stimulating Factor; NEIC: Neuron-Endocrine-Immune-Circulation;NFATc1:NuclearfactorofactivatedT-cells,cytoplasmic1;OA:Oleanolicacid;OCN: Osteocalcin; OVX: Ovariectomized; OPG: Osteoprotegerin; PLCγ2: Phospholipase Cγ2; PTH: Parathyroid hormone; RANK: Receptor activator of NF-ΚB; RANKL: Receptor activator of NF-κB ligand; Runx 2: Runt-related transcription factor 2; TCM: Traditional Chinese Medicine; TKEBP: Bushenqianggufang; TKSB: Bushengzhuanggupian;TKYDP:Bushenwenyangfang; TKYR: Bushengyangfang; LWDHP: Liuweidihuang pills; TNF-α: Tumor necrosis factor α; TRAP: Tartrate-resistant acid phosphate; YGP: Yougui Pill; ZGP: Zuogui Pill. Received 6 August 2016; Accept 8 December 2016

INTRODUCTION Zheng is the requirement for clinical physicians to perform proper treatments. Shen deficiency Zheng is a basic patholog- Tonifying Shen (Including the functions of anatomic Kidney ical identification for osteoporosis according to TCM theory and partially the function of neuro-endocrino-immune- which holds the idea that the deficiency of Shen always leading – circulation (NEIC) system) strategy works as an important to deteriorated bone structure and function[2 3]. Therefore, alternative and complementary method that is widely used Tonifying Shen strategy is intensively used in the management in Traditional Chinese Medicine (TCM) based on the proper of osteoporosis. Furthermore, Shen deficiency Zheng can be identification of Zheng. Zheng is the summarization of the divided into Shen-Yin and Shen-Yang deficiency Zheng bases complexes pathological syndromes that occur during the on osteoporosis-induced symptoms. Briefly, patients with different stages of disease(s), and both the pathogenic and flaccid waist and knees, dysphoria with smothery sensation, healthy factors are included[1]. The correct identification of and night sweat are regarded as Shen-Yin deficiency Zheng

2016|Vol. 2|Issue 4 38 www.wjtcm.org World J Tradit Chin Med 2016; 2(4): 38–48 W. Gou et al. and are treated with Tonifying Shen-Yin principle. Patients are used in treating osteoporosis. Usually, a Tonifying Shen with weak waist and knees, feeling of chilliness, and frequent prescription is constituted by many Tonifying Shen herbs micturition can be considered as the Shen-Yang deficiency and the prescription often exerts more predominant effect Zheng and are treated with Tonifying Shen-Yang principle[4]. than a single herb. Meanwhile, the effective components are Therefore, Tonifying Shen strategy is divided into Tonifying extracted from herb(s), and they are very convenient to Shen-Yin and -Yang principles. Currently, many prescriptions perform the molecular mechanistic investigations. All these are constructed to treat osteoporosis based on the exact three forms have been established to exert bone-protective identification of Shen-Yang and -Yin deficiency Zheng in TCM. effects and treat osteoporosis by the instruction of Tonifying Osteoporosis is one bone disturbance that is characterized Shen-Yin and Shen-Yang principles. However, many ques- by reduced bone mineral density (BMD) and deteriorated tions still need to be addressed: 1) the exact molecular bone structure[5]. In osteoporosis, the balanced bone remodeling mechanism of Tonifying-Shen strategy in treating osteopo- processes are disturbed by osteoclast-dominated bone resorp‐ rosis is still not fully understood. 2) Underlying mechanism – tion that overpasses osteoblast-dominated bone formation[6 7]. processes of Tonifying Shen-Yin or Shen-Yang principles in Osteoporosis always leading to the impairment of vital skeletal treating osteoporosis are still obscure. 3) Tonifying Shen-Yin functions: (a) providing support and site for the muscle to or Shen-Yang principles do exert bone-protective effects. maintain the movement, (b) protecting organs such as bone However, what is the similarity and difference between marrow and brain, and (c) acting as one metabolic organ that Tonifying Shen-Yin and Shen-Yang? Therefore, it is neces- regulatesthehomeostasisofmineralsandhormones[6].There- sary to address these important issues in search of better fore, patients who are diagnosed with osteoporosis often suffer strategies for treating osteoporosis. ‐ from skeleton pains, limitation of movement, soreness of joints, In this paper, we select the typical Tonifying Shen pre weakness of the waist and knees and so on[8]. According to scriptions (herbs, effective components) which play bone- the theory of TCM, osteoporosis-induced syndromes can be protective to address the following points: 1) first explore the regarded as the Shen-Yin or Shen-Yang deficiency Zheng[4] bone-protective effects and molecular mechanisms of Tonify- (Fig. 1). However, the mechanism by which Tonifying Shen ing Shen strategy, particular their effects in regulating the strategy, particular two principles of Shen-Yang and Shen-Yin differentiation of MSCs into osteoblasts, and the differentia- principles, exerts bone-protective effects in regulating bone tion of HSCs into osteoclast. 2) we further explore the bone- remodeling are still rarely investigated. protective effects and molecular mechanisms of Tonifying Shen-Yang and -Yin principles in treating osteoporosis are Osteoporosis reflects the imbalance of bone formation by systematically analyzed. 3) we explore the similarities between osteoblast and bone resorption by osteoclast in favor of bone Tonifying Shen-Yang and -Yin principles in treating osteopo- resorption[5,7]. Therefore, drugs that inhibit osteoclast formation/ rosis are identified. 4) more important, the difference between activity or stimulate osteoblast formation/activity are considered – Tonifying Shen-Yin and Shen-Yang principles in treating as effective methods for treating osteoporosis[9 13]. However, osteoporosis are established. currently marketed drugs in treating osteoporosis have caused – We hope that this paper will not only help gain new heavy economic burdens or side effects[14 16].TonifyingShen insights into Tonifying Shen-Yin or Shen-Yang in treating strategy, including Tonifying Shen-Yin or Shen-Yang prin- osteoporosis, but also broaden our strategies in the treatment ciple, has been established to exert bone-protective effects of osteoporosis. In addition, our findings will promote new as well as rescuing bone loss in osteoporosis patients and – drug discoveries in treating osteoporosis. in vivo investigations[17 19]. Till now, TCM prescriptions, herbs and effective components are three mainly forms that 1 TONIFYING SHEN STRATEGY IN TREATING OSTEOPOROSIS 1.1 Clinical investigations In the late 1980s, clinical studies observed the bone-protective effects of Tonifying-Shen prescriptions and herbs in treating – patients who were diagnosed with osteoporosis[18 20]. One clinical investigation reported that Bushenfang (BSF, the ingredients are seen in the Table1/2) in TCM both improved osteoporosis-induced symptoms and delayed the developing process of osteoporosis[18]. Furthermore, one subsequent study Figure 1. Shen-Yin deficiency and Shen-Yang deficiency Zheng in found that Tonifying Shen prescription named Bushenjiangu- osteoporosis. A. Patient who were diagnosed with osteoporosis that can fang (BSJG, table 1&2) capsule might alleviate bone loss be identified as the Shen-Yin deficiency Zheng. B. Patient who were and symptoms in postmenopausal osteoporosis[19]. This study fi diagnosed with osteoporosis that could be identi ed as the Shen-Yin found that the syndromes in nearly 90% osteoporosis patients deficiency Zheng. C. Patient who were diagnosed with osteoporosis that can be considered as both the Shen-Yin and -Yang deficiency Zheng. (Totally 51 cases) treated with BSJG capsule for 3 months D. Patient who were diagnosed with osteoporosis but not regarded as the were improved in comparison with calcium group (P<0.05). Shen-Yin or Shen-Yang deficiency Zheng in TCM. Moreover, BMD of the lumbar vertebra in 68.8% patients was

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Table 1. Formulae for Tonifying Shen strategy

Tonifying Shen & Formula Ingredients Yin & Yang category References

BGSSC: 6 herbs: yín yáng huò (Epimedii Herba); buˇ guˇ zh¯ll (Psoralea cory lifolia L); Shen [51] Bugushensuipian sheng huáng qí (Astragali Radix Cruda), gu¯ball ˇn (tortoise plastron); bie¯ jiaˇ (turtle shell), da¯ng gull (Angelicae Sinensis Radix), etc. BSF: 5 herbs: dù zhòng (Eucommiae Cortex); lù róng Shen [18] Bushenfang: (Cervi Cornu Pantotrichum); yín yáng huò (Epimedii Herba), buˇ guˇ zh¯ll (Psoralea corylifolia L); Dì Huáng (Rehmannia glutinosa), etc. BSJG: 8 herbs: Dì Huáng (Rehmannia glutinosa); buˇ guˇ zh¯ll Shen [19] Bushenjiangufang (Psoralea corylifolia L; goˇuqˇll zˇll (Wolfberry Fruit); dù zhòng (Eucommiae Cortex), sha¯n yào (Dioscoreae Rhizoma); sha¯n zhu¯ yú (Fructus corni); lù jiaˇo (Deer horn); da¯ng gu¯ll (Angelica sinensis), etc. BSSF: 6 herbs: goˇuqˇll zˇll (Wolfberry Fruit); Dì Huáng (Rehmannia glut inosa); Shen [21–23] Bushenshenggufang buˇ guˇ zh¯ll (Psoralea corylifolia L; niú x¯ll (Achyranthes bidentata); gu¯ball ˇn (tortoise plastron); tù s¯zll ˇı (semencuscutae), etc. BSYG: 6 herbs: gu¯ball ˇn (tortoise plastron); bie¯jiaˇ (turtle shell); Shen [49] Bushengyanggupian yín yáng huò (Epimedii Herba); buˇ guˇ zh¯ll (Psoralea corylifolia L); dà huáng (Rhei Radix et Rhizoma); sha¯n yào (Dioscoreae Rhizoma), etc. IKSBC: 8 herbs: Dì Huáng (Rehmannia glutinosa); buˇ guˇ zh¯ll Shen [29] Bushenjiangujiaonang (Psoralea corylifolia L; goˇuqˇll zˇll (Wolfberry Fruit); dù zhòng (Eucommiae Cortex), sha¯n yào (Dioscoreae Rhizoma); sha¯n zhu¯ yú (Fructus corni); lù jiaˇo (Deer horn); da¯ng gu¯ll (Angelica sinensis), etc. TKEBP: 5 herbs: gu¯ball ˇn (tortoise plastron); bie¯jiaˇ (turtle shell); Shen [20] Bushenqianggufang yín yáng huò (Epimedii Herba); buˇ guˇ zh¯ll (Psoralea corylifolia L); dà huáng (Rhei Radix et Rhizoma), etc. largely increased in contrast to calcium supplementation after 1.2 Mechanism of Tonifying-Shen strategy in the treatment for 6 months. Mechanistic findings indicated treating osteoporosis that the increased bone formation and depressed bone The coupling mechanism in bone remodeling between the absorption were attributed to bone-protective effect[19].In osteoblast-mediated bone formation and osteoclast-mediated addition, one clinical study with 52 osteoporosis patients was bone resorption ensures no net changes of bone mass and performed to investigate the bone-protective effects of Bush- bone strengthen. Osteoblasts are differentiated from MSCs enqianggufang (TKEBP, table1&2). After the treatment for 3 by the stimulation of necessary cytokines, and the functions months, patients treated with TKEBP showed significant of osteoblast determine the ratio of new bone formation[6]. higher levels of blood alkaline phosphates (ALP), beta 2- Osteoclasts are differentiated from HSCs through the stimula- microglobulin (beta 2-MG) level, urinary beta 2-MG, calcium tion of macrophage colony Stimulating factor (M-CSF) and and phosphorus. Moreover, the symptoms of lumbar dorsal receptor activator of NF-κB ligand (RANKL)[7]. In pathologi- pain, general fatigue, palpitation and vertigo were improved cal conditions, the over-activated osteoclast causes more bone after the treatment[20]. Taken together, these studies estab- resorption than overpass bone formation, leading to the lished that Tonifying Shen strategy is effective in preventing occurrence of osteoporosis[5]. Many pathological factors bone loss and alleviating osteoporosis-induced symptoms. contribute to the occurrence of osteoporosis by causing the From then on, series of studies were carried out with larger imbalance the formation/activity of osteoblast and osteoclast. numbers of patients, and more representative bone biological Till now, Tonifying-Shen strategy, including prescriptions/ – markers in osteoporosis[21 22]. These studies confirmed that herbs/effective components, has been proved to exert bone- Tonifying Shen prescriptions/herbs administration significantly protective effects through many aspects in affecting bone increased BMD and relieved the symptoms in treating osteopo- biology. rosis[21]. Mechanistically, one study indicates the expressions of bone gla protein (BGP), pyridinoline, estradiol, testosterone, and 1.2.1 Bone mineral homeostasis blood urea nitrogen were greatly increased after the treatment In the 1940/50s, scientists have realized that normal bone with Bushenshenggufang (BSSF, table1/2)[22]. Moreover, one is composed of different crystal minerals[24] and two most of Five-year-follow-up study observed the long-term bone-protec- them are calcium and phosphorus. Subsequent investigations tive effects with BSSF[23]. 194 patients participated, and the indicated that nearly 99% calcium and 84% phosphorus are results showed that BSSF greatly increased BMD from baseline deposited as crystals in bone. Therefore, calcium and phos- (0.211 ± 0.022 g/cm) at the end of (5 year) the study (0.284 ± phorus are two critical minerals in determining bone structure – 0.015 g/cm), whereas the control group decreased significantly and functions[25 27]. The regulatory effects of Tonifying Shen from baseline (0.212 ± 0.023 g/cm) to one higher level (0.187 ± strategy in treating osteoporosis by regulating minerals 0.022 g/cm). homeostasis were investigated later on.

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Table 2. Formulae for Tonifying Shen strategy

Tonifying Shen & Formula Ingredients Yin & Yang category References

TKSB: 6 herbs: goˇuqˇll zˇll (Wolfberry Fruit); Dì Huáng (Rehmannia glutinosa); Shen [38,39] Bushengzuanggupian sha¯n yào (Dioscoreae Rhizoma); niú x¯ll (Achyranthes bidentata); gu¯ball ˇn (tortoise plastron); tù s¯zll ˇll (semen cuscutae) TKYDP: 7 herbs: lù róng (Cervi Cornu Pantotrichum); Yang [55] Bushenwenyangfang yín yáng huò (Epimedii Herba), goˇuqˇll zˇll (Wolfberry Fruit); Dì Huáng (Rehmannia glutinosa); sha¯ n yào (Dioscoreae Rhizoma); niú x¯ll (Achyranthes bidentata); gu¯ball ˇn (tortoise plastron), etc. TKYR: 7 herbs: goˇuqˇll zˇll (Wolfberry Fruit); lù róng Yang [54] Bushengyangfang (Cervi Cornu Pantotrichum); yín yàng huò (Epimedii Herba), Dì Huáng (Rehmannia glutinosa); sha¯ n yào (Dioscoreae Rhizoma); niú x¯ll (Achyranthes bidentata); gu¯ball ˇn (tortoise plastron), etc. GLP: 6 herbs: goˇuqˇll zˇll (Wolfberry Fruit); Dì Huáng Shen [17,33] Gulin Pill (Rehmannia glutinosa); sha¯n yào (Dioscoreae Rhizoma); niú x¯ll (Achyranthes bidentata); Huáng qí (Astragali Radix), tù s¯zll ˇll (semen cuscutae), etc. ZGP: 8 herbs: Dì Huáng (Rhemannia glutinosa); sha¯n zhu¯ yú (Fructus Yin [4] Zuogui Pill corni); sha¯n yào (Dioscoreae Rhizoma); goˇuqˇll zˇll (Wolfberry Fruit); niú x¯ll (Achyranthes bidentata); lù jiaˇo (Deer horn); gu¯ball ˇn (tortoise plastron); tù s¯zll ˇll (semen cuscutae), etc. YGP: 8 herbs: Dì Huáng (Rhemannia glutinosa); dàn fù piàn (Aconiti Yang [4] Yuogui Pill Radix Lateralis Insalsa); ròu guì (cortex cinnamomi); tù s¯zll ˇll (semen cuscutae); goˇuqˇll zˇll (Wolfberry Fruit); dù zhòng (Eucommiae Cortex), sha¯n yào (Dioscoreae Rhizoma); sha¯n zhu¯ yú (Fructus corni); lù jiaˇo (Deer horn); da¯nggu¯ll (Angelica sinensis), etc. LWDHP: Dì Huáng (Rehmannia glutinosa); sha¯ n zhu¯ yú (Fructus Yin [65] Liuweidihuang pills corni); fú líng (Poria cocos); sha¯n yào (Dioscoreae Rhizoma); zé xiè (Alismatis Rhizoma), muˇ da¯n pí (Tree Peony Bark), etc.

One in vivo study evaluated the bone-protective effect prescription, Gulin Pill (GLP), has been indicated exerting of Fructus Ligustri Lucidi (FLL), a Tonifying Shen-Yin herb, bone-protective effects by stimulating osteoblast differentia- in ovariectomized (OVX) rat. The result showed that FLL tion in MG-63 cells. The mechanistic investigation indicated could reverse the OVX-induced bone loss and increase BMD. that GLP treatment significantly promotes the proliferation Particularly, FLL intervention alleviated OVX-induced calci- and differentiation of MG-63 cells by stimulating the expres- um loss in rats by increasing intestinal calcium absorption sion of OPG, and down-regulating the expression of RANKL 2+ and suppressing urinary Ca excretion. Further mechanistic at the protein and mRNA levels. Furthermore, GLP-treated investigations established that FLL could modulate bone remodeling by affecting calcium homeostasis in OVX rat[28]. Subsequently studies with one Tonifying Shen prescription named Bushenjiangujiaonang (IKSBC, table1&2) showed that IKSBC attenuated bone loss in OVX-induced osteoporosis rats. The concentrations of calcium and phosphorus in serum from OVX rat was increased after treatment with IKSBC. Meanwhile, the expression of ALP was increased, and the trabecular bone volume was significantly increased in contrast to the pretreatment[29]. Therefore, the bone-protective effect Figure 2. (A) Mechanism of Tonifiying Shen strategy in regulating of Tonifying Shen strategy may partly be due to their osteoblastogenesis. During normal osteoblastogenesis, Tonifiying Shen regulatory effects on the homeostasis of calcium and phos- strategy, including Tonifiying Shen prescription, herbs, effective compo- phorus (Fig. 2A&2B). nents, stimulates the expression of estrogen signaling, Wnt/β-catenin, Vitamin D, Notch, and BMPs to stimulate osteoclastogenesis. Meanwhile, they play bidirectional effects on osteoprotegerin (OPG) and regulating 1.2.2 RANKL/RANK/OPG axis osteoblastogenesis. In addition, they also regulated the transportation of RANKL/RANK/OPG (Osteoprotegerin) axis was discovered calcium and phosphors that necessary for normal osteoblastogenesis. in the late 1990s, and has been shown to play a critical role in (B) Mechanism of Tonifiying Shen strategy in controlling osteoclastogenesis. fi coordinating bone remodeling. Both osteoblast and osteoclast During osteoclastogenesis, Tonifying Shen strategy, including Toni ying [30–32] Shen prescriptions, herbs, effective components, stimulate the expression are regulated by this axis , and studies have shown that of estrogen signaling down-regulated interlunkin-1, 6, 7, TNFs and Tonifying Shen strategy may regulate bone remodeling by RANKL, osteoclastogenesis and the transportation of calcium and affecting RANKL/RANK/OPG axis. One Tonifying Shen phosphors.

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Wnt/β-catenin and BMP signaling. This finding indicates that osthole may be one potential to stimulate new bone formation to prevent estrogen deficiency-induced bone loss (Fig. 2A&2B). Psoralen, one effective component from Tonifying-Shen herb, was found to promote osteoblast differentiation by up- regulating the expressions of osteoblast-specific genes, including type I collagen (Col-1), osteocalcin (OCN), bone sialoprotein (BSA). One further study indicated that psoralen stimulates the expression of BMP-2 and BMP-4, and increase Figure 3. One schematic illustration of Tonifying Shen strategy in the level of phospho-Smad1/5/8 as well as the expression of [44] regulating bone homeostasis. Tonifying Shen strategy, including prescrip- osterix . tions, herbs, effective components, stimulates the mensenchymal stem cells (MSCs) into osteoblast. B. Tonifying Shen strategy, suppress the 1.2.5 Notch signaling MSCs into adipocytes. C. Tonifying Shen strategy suppress hemateopoeitic Notch signaling is necessary for the differentiation of stem cell (HSCs) into osteoclast. osteoblasts by working together with BMP, and Wnt[45], and is considered as one important factor for osteoblast differen- serum suppressed the RANKL-activated expression of p38, [45–46] one important signaling molecule for osteoclast differ‐ tiation in MSCs . Oleanolic acid (OA) and its glycosides entiation. This finding indicates that GLP may effectively have been reported to prevent bone loss in OVX-induced promote the proliferation and differentiation of osteoblasts by mice according to Micro-CT assessment. Subsequently, one regulating the RANK/RANKL/OPG ratio and the expression animal investigation indicated that OA-treated (10mg/kg/day, of p38 (Fig.2A&2B)[17]. RANKL plays critical roles in regulat- ip.) mice displayed increased osteoblast numbers, higher ing osteoclast differentiation and function, we therefore expression of OCN and Runt-related transcription factor propose that the suppressed bone resorption may contribute 2 (Runx2). Furthermore, in vitro studies indicated that OA to the bone-protective effects of Tonifying Shen strategy in exerts an osteoprotective effect in OVX-induced osteoporotic treating osteoporosis[33]. mice by enhancing osteoblastic differentiation of MSCs. Molecular investigations indicated that this effect was due to [49] 1.2.3 Estrogen signaling their regulatory effect on Notch signaling (Fig. 2A&2B) . The decreased level of estrogen causes enhanced osteoclast- induced bone resorption by increasing the production cyto- 1.2.6 Vitamin D3 Vitamin D3 and its active ingredients supplementation, 1, kines that are necessary for osteoclast formation and/or activity, 25-dihydroxy Vitamin D3 (1, 25(OH) D3), have been reported such as interleukin (IL)-1,6,7, tumor necrosis factor (TNF-α) [34–37] to play critical roles in determining the bone formation and and RANKL . Therefore, estrogen deficiency is a major structure[48]. One animal study discovered that the level of 1, pathologic factor in bone loss after menopause. One previous 25(OH) D3 in blood serum, liver and Kidney was significantly study identified the bone-protective effects of the prescription elevated after the treatment of Bushengyanggupian (BSYG, named Bushengzhuanggupian (TKSB, table1&2) in the OVX- table1&2) in OVX-induced osteoporotic rats. Meanwhile, induced (Lower level of estrogen model) rats. As the results BSYG-treated group rats showed an increase of BMD in showed, the activity of tartrate-resistant acid phosphate (TRAP) femoral heads compared with saline control. Moreover, bone was remarkably suppressed after the intervention with TKSB. maximal load and stress index was improved after the Meanwhile, TRAP activity in higher and lower dosage of TKSB intervention of BSYG[49]. These studies indicate that Tonify- were both decreased[38]. This study indicated that TKSB may ing Shen prescriptions/herbs might stimulate bone forma‐ exert the estrogen-like effect on bone biology (Fig. 2B&2C)[39]. tion through up-regulating the level of 1, 25(OH) 2D3 Furthermore, studies have shown that estrogen deprivation may (Fig. 2A&2B). be one of the potential factor to induce the Shen-Yin deficiency Zheng model in TCM[40], which may be one future direction for further investigations (Fig. 2A&2B). 1.3 Tonifying Shen strategy in regulating homeostasis 1.2.4 Wnt/β-catenin/ BMP signaling Therefore, Tonifying Shen strategy, including the applica- Wnt/β-catenin and bone morphogenetic protein (BMP) tions of Tonifying Shen prescriptions, herbs and effective works cooperatively to promote osteoblast differentiations of components, exerts bone-protective effects in osteoporosis by – MSCs[41 42] by activating series of osteoblast differentiation affecting bone formation and resorption. At the cellular level, markers. The intervention of osthole, a coumarin-like deriva- they exert bone-protective effects by promoting MSCs into tive extracted from one Tonifying Shen herb, was found to be osteoblast and bone formation (Fig. 3). Meanwhile, Tonifying able to increase new bone formation in OVX-induced bone Shen strategy may also promote MSCs into more osteoblasts loss in rats. In vitro studies demonstrated that osthole activate instead of adipocytes[47]. On the other hand, Tonifying Shen the expression of Wnt/β-catenin and BMP-2 to enhance new strategy has been established to exert inhibitory effect on bone formation[43]. Moreover, osthole exerted the stimulatory HSCs-originated osteoclastogenesis and bone resorption[50]. effect on promoting osteoblast differentiation through activating Next, further studies have been performed to investigate

2016|Vol. 2|Issue 4 42 www.wjtcm.org World J Tradit Chin Med 2016; 2(4): 38–48 W. Gou et al. the underlying processes of how these two principles in Meanwhile, gene profiles screen revealed a significant shift of Tonifying Shen strategy, Tonifying Shen-Yin and -Yang, in osteoblast-related gene expression after the intervention of treating osteoporosis. icariin, which were more significance in CORT-treated rat[53]. Dexamethasone (DM) was always used to induce Shen-Yang 2 TONIFYING SHEN-YANG PRINCIPLES IN deficiency Zheng model in animals. One study investigated the efficacy of Bushengyangfang (TKYR, table1&2) in osteo‐ TREATING OSTEOPOROSIS porotic rats with DM injection. In TKYR treated group, 2.1 Clinical Studies the BMD, femur bending strength, serum-BGP, serum-PTH More than 20 years ago, Tonifying Shen-Yang principle and serum-E2 in the TKYR-treated group were significantly has been selected in the management of osteoporosis with higher than DM-treated group (P<0.01). Meanwhile, com‐ Shen-Yang deficiency Zheng in TCM to investigate the pared with DM group, the levels of serum-T in TKYR bone-protective effect and the mechanisms[51]. One Tonifying prevention or TKYR treatment group were significant [54] Shen-Yang prescription, Bugushengsui capsule (BGSSC), was increased (P<0.05) . One investigation established the investigated in treating primary osteoporosis. The clinical underlying mechanism of the Bushenwenyangfang (TKYDP, effects in patients treated with BGSSC were significantly higher table1&2) in regulating osteoblast formation/activity. Human than control (Vitamin D plus calcium). Moreover, BGSSC osteoblastic cell line (hFOB 1.19) was treated with 10% intervention improved BMD according to the test with double TKYDP administrated pattern-serum from postmenopausal energy X-ray as well as increasing the level of serum calcitonin, women (30 cases, aged 60-70). The cell viability, expression of hormone and calcium. This clinical finding indicates that the ALP and numbers of calcified nodules in TKYDP treated- bone-protective effects of BGSSC on primary osteoporosis serum were remarkably increased in hFOB1.19 cells as well including the improvement of BMD, and suppressed effect as decreasing the expression of RANKL. In addition, the on bone resorption. Moreover, one randomized, double- concentration of estradiol (E2), and insulin-like factor-1 (IGF- blind, multicenter, and placebo-controlled study revealed 1) in the TKYDP treated-serum was higher in patients than that Tonifying Shen-Yang prescription, Yougui Pill (YGP, calcium control. Therefore, these findings indicate that the table1&2), may largely relieve the syndromes in patients with down-regulation of estradiol E2, IGF-1, RANKL in TKYD- osteoporosis. Moreover, a total of 200 patients, aged from treated serum may contribute to the increased bone formation [55] 55-75, diagnosed with lower BMD were enrolled. Moreover, as well as decreasing bone resorption in patients . YGP may significantly relieve the osteoporosis related syn- Osthole is one effective component from Fructus Cnidii, dromes, such as weak waist/knee, and extreme chilliness[4]. a traditional herb of Tonifying Shen-Yang (Table 3). One investigation indicated that osthole could promote the activity 2.2 Mechanism of Tonifying Shen-Yang principle of ALP and the formation of bone nodules by enhancing in treating osteoporosis osteoblastic proliferation and differentiation[56]. Moreover, Animal models with Shen-Yang deficiency Zheng and osthole intervention can greatly prevent OVX caused bone osteoporosis phenotype were successfully investigated by the loss in rats by improving bone micro architecture. Further usage of the high dose of corticosteroids (CORT) in vivo (52). molecular assays demostrated that osthole activated the Shen-Yang deficiency zheng with osteoporosis was treated expression of Wnt/β-catenin and BMP-2[43]. On the other with Tonifying Shen-Yang herbs, effective component from hand, following studies indicated that osthole inhibited osteo- Tonifying Shen-Yang prescription or herbs to assess their clast formation and bone resorption pits on bone slices. bone-protective effect. Icariin is an active component from Further mechanistic studies indicated that osthole inhibited Herbal Epimedium, one Tonifying Shen-Yang herb in TCM. the activity of osteoclast and bone resorption through suppres- One investigation induced osteoporosis rats by CORT sing the RANKL/TRAF6/Mkk/JNK signaling cascade[57]. injection and OVX, and both were treated with icariin for 2 In addition, one study found that icariin dose-dependently weeks and 3 months. This result indicated that icariin may inhibited the growth and differentiation of HSCs, and less promote MSC differentiation from CORT and OVX-induced TRAP positive cells formation were observed in icariin-treated rat by stimulating the secretion of OCN, Col-1, and Runx2. group. Meanwhile, bone resorbed pits formation was also

Table 3. Efffective components from Tonifying Shenn strategy

Tonifying Shen & Effective component Herbs Yin & Yang category References

Oleanolic acid mò hàn lián (Yerbadetajo Herb) Yin [47,66–68] ˇ nü zhe¯n zˇll (Fructus Ligustri Lucidi) Icariin yín yáng huò (Herbal Epimedium; Yang [43,50,53,58,59,60] Epimedium koreanum Nakai) Osthole shé chuáng zˇll (Fructus Cnidii) Yang [43,56,57] Psoralen buˇ guˇ zh¯ll (Psoralea corylifolia L) Yang [44,69–71] ˇ nü zhe¯n zˇll (Fructus Ligustri Lucidi) Yin [28] Dì Huáng (Rehmannia glutinosa) Yin [64]

www.wjtcm.org 43 2016|Vol. 2|Issue 4 © World Journal of Traditional Chinese Medicine Tonifying Shen-Yin and -Yang in osteoporosis dramatically decreased than saline control. RT-PCR analysis Subsequently, one study indicated that FLL enhanced BMD indicated that the gene expression of TRAP and RANK and mechanical properties in growing male rats[63]. Mechanistic was greatly down-regulated in bone marrow macrophages data demonstrated that FLL could alter bone turnover by (BMMs)[50,58]. Moreover, the study found that icariin reduced increasing bone formation biomarkers, such as the expression the size of lipopolysaccharide (LPS)-induced osteoclast forma- of OCN and CTX-I, and both calcium absorption and retention tion, and the expression of TRAP. On the other hand, icariin rates were elevated after FLL treatment. Mechanistic studies inhibited ALP activity without inhibiting cell viability. Mean- indicated that the expression of 1α-hydroxylase, duodenum, while, icariin also inhibited LPS-induced bone resorption and and calcium absorption-related genes were increased to activate the expression of IL-6 and TNF-α in BMMs. Gene expression the 1, 25(OH) 2D3-dependent calcium transportation in stimu‐ of OPG was up-regulated, and the expression of RANKL was lating bone formation. suppressed. Mechanistic findings indicated that icariin inhib- Dried root of rehmanniaglutinosa (DRG) is a Tonifying ited LPS-induced osteoclast formation by suppressing the Shen-Yin herb in TCM for the treatment of osteoporosis. One [43] expression of p38 and JNK . Later on, one study finding study indicated that treatment with DRG caused the decrease of indicated that bone loss was attenuated by the treatment with BMD in the femur and lumbar in OVX-induced bone loss in [59] icariin in OVX-induced osteoporotic rats . In addition, mice without affecting the body, organs, and uterus weights. icariin intervention could completely correct the decreased Further mechanistic investigation indicates that serum ALP serum concentration of calcium, and phosphorus in OVX rats, level in the DRG-treated group was increased. This study and biomechanical strength in icariin treated rats was also indicated that DRG is able to prevent the OVX-induced bone largely enhanced in comparison to sham Moreover, icariin [64] loss in influencing the level hormones, such as estrogen . intervention could stimulate new bone formation in OPG knockout mice after local injection in calvarias, and reversed OPG Knockout-induced bone loss. Mechanistic assessments 3.2 Mechanism of Tonifying Shen-Yin indicated that icariin up-regulated the expression of BMP-2, principle in treating osteoporosis BMP-4, Runx2, OC, Wnt1, and Wnt3a in OPG Knockout The Shen-Yin deficiency Zheng with osteoporosis animal mice. Furthermore, icariin intervention increased the expres- models were successfully established by steroid intervention, sion of Axin2, DKK1, TCF1, and LEF1, which are the direct such as estrogen deprivation[65]. Compared with control group, targeted genes of β-catenin signaling pathway. These findings mice with Shen-Yin deficiency (Estrogen deprivation) group indicated that icariin reversed the phenotypes of OPG- appeared series of symptoms, including hair dry, restlessness, deficient mice through enhancing the activation of Wnt/β- excitability and hard stool. This Shen-Yin deficiency Zheng catenin or BMP signaling[60]. In vitro assays found that icariin model is stable and reliable for the integration of disease and promoted MC3T3-E1 osteoblastic differentiation and miner- syndrome in TCM[65]. Molecular studies have shown that the alization by enhancing the expression of ALP, Col-I, and bone level of ACTH, cAMP, cGMP and TSH in serum was greatly nodule formation. Molecular investigations indicated that the decreased in this Shen-Yin deficiency Zheng model. Mean- expression of mitogen-activated protein kinase (MAPK) - while, BMD of lumbar vertebra and femur was decreased activated signaling, such as ERK and JNK, contributed to the according to HE staining. However, Liuweidihuang pills [61] stimulatory effect of icariin on osteoblastogenesis . DM has (LWDHP, table1&2), one famous Tonifying Shen-Yin pre- been routinely used as a classical inducer for osteoblastogen- scription, could partly reverse the bone loss phenotype and as ‐ esis but one investigation indicated that icariin interven well as Alleviating the level of ACTH, cAMP, cGMP, and TSH. tion stimulate one higher expression of ALP activity, OCN OA, one effective component from Tonifying Shen-Yin secretion, larger mineralized nodules, and calcium deposition, herb (Table 3), has been established to exert bone-protective higher levels of mRNA expression of osterix, Runx2, and Col- effects by inhibiting the formation of osteoclast, and bone α [62] 1 in contrast to the DM-treated group . resorptive activity of osteoclast in RAW264.7 cell line[66]. Meanwhile, OA selectively induced the apoptosis at an early stage of mature osteoclast via increasing the caspase-3 activity. 3 TONIFYING SHEN-YIN PRINCIPLES IN Furthermore, OA intervention significantly prevented bone TREATING OSTEOPOROSIS loss in OVX mice without any hormone-like adverse effects, 3.1 Clinical Studies and different OA derivatives may exert different effects on [67] One clinical study indicated that the administrations of ZGP, osteoclast . One molecular investigation indicated that OA one famous Tonifying Shen-Yin prescription, did not signifi- inhibited RANKL-induced osteoclast formation by suppres- cantly relieve the symptoms of Shen-Yin deficiency Zheng in sing the phosphorylated expression of Btk and phospholipase 2+ patients who were diagnosed with osteoporosis. However, the Cγ2 (PLCγ2), calcium (Ca ) oscillation, and the expression results indicated that ZGP intervention remarkably improved of nuclear factor of activated T-cells, cytoplasm 1 (NFATc1). BMD at lumbar spine than placebo after the treatment for Furthermore, OA-intervention attenuated LPS-induced bone 6 months. Moreover, BMD in the lumbar spine of ZGP group erosion based on μ-CT and histological analysis in femur was increased by 4.1% and 4.7% after another 6 months in vivo[68] as well as rescuing OVX-induced osteoporosis rat follow-up study[4]. Another clinical investigation found that model via stimulating the differentiation of bone MSCs by FLL could alleviate bone loss in osteoporosis patients. suppressing Notch signaling[47].

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Psoralen, one effective component from Tonifying Shen- Yin herb (Table 3), has been reported to exert bone-protective effects. Psoralen-treated osteoblastogenesis and ultra-structural assessment showed that the effect of psoralen on osteoblast formation ranged from negligible to unambiguous structural alterations[69]. One animal investigation indicated that there were more new bone presented after the treatment with psoralen than the normal control (4.5:1). Meanwhile, cell culture assays indicated that the osteoblast number was also predominantly increased in the psoralen-treated group[70]. Subsequently, investigations indicated that psoralen dose- Figure 4. The comparison of Tonifying Shen-Yin and Shen-Yang principles dependently promoted osteoblast differentiation from pri- in treating osteoporosis. Tonifying Shen-Yang and Shen-Yin both plays bone-protective effect by increasing osteoblast-determined bone forma- mary mouse calvarias. The mechanistic assessment indicated tion and inhibiting osteoblast-dominated bone resorption. However, that the stimulatory effects may be due to enhanced Tonifying Shen-Yin principle mainly increases bone mineral density (BMD) [44] expressions of osteoblast-specific marker genes . Interest- and prevents bone loss. Tonifying Shen-Yang mainly exerts effects on ingly, osteoblastogenesis assessment indicated that the osteoclast-dominant bone resorption and relieving osteoporosis-caused intervention of psoralen elevated the expression of ALP in symptoms. osteoporotic rats, and the formation of unit osteoblast[71]. For this study, 200 patients who were diagnosed with 4 COMPARISON OF TONIFYING SHEN-YIN osteoporosis aged from 55-75, were enrolled. All patients AND -YANG PRINCIPLE IN TREATING were treated with ZGP or YGP after the proper identification of Shen-Yin or -Yang deficiency Zheng. Patients with Shen- OSTEOPOROSIS Yang deficiency Zheng were treated with YGP, while those Current studies have established the common mechanisms with Shen-Yin deficiency Zheng were treated with ZGP. of Tonifying Shen-Yin and Shen-Yang principles in treating The result showed that YGP significantly reduced pains osteoporosis, and both bone formation and bone resorption according to VSA and ECOS-16 questionnaire scores than were affected. Molecular investigations have demonstrated placebo at 9, and 12 months. ZGP could significantly increase that a variety of signaling pathways in osteoblastogenesis BMD in lumbar spine in patients. Therefore, ZGP significant- and osteoclastogenesis were involved in. In order to clearly ly increased lumbar spine BMD, whereas YGP significantly address the similarity as well as the difference between these reduced symptoms such as the severe of pain, and improved two principles, we compared the effects of Tonifying Shen- the life quality in patients[4]. Yin and -Yang principles in treating osteoporosis as the bellowed: (1) Tonifying Shen-Yin and -Yang principles both are 5 DISCUSSIONS effective methods for treating osteoporosis, such as Shen deficiency is the pathogenesis in osteoporosis in TCM, increasing BMD, alleviating osteoporosis-induced symp- and Tonifying Shen strategy is the basic rule in the manage- toms, and modulating series of bone metabolic markers. [8] Mechanistic studies indicated that Tonifying Shen-Yin or ment of osteoporosis . Currently, Tonifying Shen prescrip- -Yang principle exerts stimulatory effects on enhancing tions/herbs are the most common agents used in treating [8,39,72] osteoblast differentiation/activity and bone formation. osteoporosis in clinic practice of TCM . One study On the other hand, both principles exert inhibitory reviewed that herbal prescriptions have been used in treating effects on osteoclast differentiation/activity and bone osteoporosis throughout TCM history. Totally 389 prescrip- resorption (Fig. 4). tions were found, and 238 herbs were involved with a total (2) Tonifying Shen-Yin principle exerts the favorable effect frequency of appearance at 4236. According to the result, on increasing BMD by stimulating the activity of Tonifying Shen herbs are the most commonly used in treating osteoblast and bone formation. Therefore, the bone- [72] protective effect of Tonifying Shen-Yin principle exerts osteoporosis . Furthermore, the most common categories bone-protective effects due to their stimulatory effect were Tonifying Shen-Yang medicinal, and nearly account for on osteoblast-mediated bone formation and inhibitory 37.8% of the total frequency. Therefore, a proper selection of effect on bone resorption (Osteoclast) in favor of the Tonifying Shen-Yang and -Yin principles with proper herbs/ former (Fig. 4). prescriptions is necessary for the treatment of osteoporosis (3) Tonifying Shen-Yang exerts a more remarkable effect in order to obtain better clinic outcomes[73]. Although both on alleviating the osteoporosis-induced symptoms. Tonifying Shen-Yin and -Yang principles are effective in treat‐ Therefore, this effect is mainly due to their preferable ing osteoporosis, but underlying process are quite different. inhibitory effects on bone resorption (Osteoclast) and stimu- Our finding first implies that Tonifying Shen-Yin principle latory effect on bone formation (Osteoblast) in favor of the significantly increased BMD in bone. In contrast, Tonifying former (Fig. 4). This finding is in consistent with results of one Shen-Yang principle largely improved osteoporosis related previous clinical investigation carried out with ZGP and YGP[4]. symptoms, and life qualities.

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Bone remodeling is maintained through the coupling and -Yin principle on NECI axis in osteoporosis. 4) As mechanisms between bone formation and bone resorption. Tonifying Shen-Yang and Shen-Yin principles exert bone- Therefore, the purpose of osteoporosis treatment is to restore protective effects simultaneously by affecting different signaling the balance of bone formation and bone resorption. Therefore, pathways during osteoblastogenesis and osteoclastogenesis, it is the coupling mechanisms between osteoblast formation/activi- necessary to examine the cross-talk signaling pathways. There- ty and osteoclast formation/activity represent two independent fore, future research need to focus on in gaining more insights but united aspects in bone remodeling. Given previous into the molecular mechanism of Tonifying Shen-Yin or -Yang investigations have proposed that osteoblast mediated-bone principles in treating osteoporosis. formation and osteoclast-mediated bone resorption should be regarded as Shen-Yin and -Yang in bone[2]. Therefore, the COMPETING INTERESTS pathogenesis of Shen-Yin deficiency Zheng in osteoporosis is mainly accompanied by impaired osteoblast-determined bone All the authors state that they have no conflicts of interest formation, whereas Shen-Yang deficiency Zheng is mainly due or disclosure. to more osteoclast activated bone resorption[74]. More recent studies have proved that osteoclast activated bone resorption ACKNOWLEDGMENTS (Yang) are the main cause of pains in osteoporosis[75]. There‐ fore, the inhibitory effect of Tonifying Shen-Yang principle on Research reported in this publication was supported by grants osteoclasts (Bone resorption) may account for clinical effects of from National natural science of Youth fund (81300568), alleviating osteoporosis-induced pains. On the other hand, Shanghai Yangfan Program (15YF1412200), the Independent Tonifying Shen-Yin principle may increase BMD by exerting Innovation Program Supported by Science and Technology “ ” ‐ the stimulatory effect on osteoblastogenesis and bone forma- Innovation of the 085 First-class Disciplines Construc tion (Yin). tion of Shanghai University of TCM (085ZY1217), and the Targeting osteoclasts and osteoblasts have long been regarded National Natural Science foundation of major international as critical strategies not only in treating osteoporosis, but also cooperation projects (81220108027). – many other diseases with osteoporotic phenotypes[76 78], such as diabetes, osteoarthritis, and cancers induced bone REFERENCES metastasis. Both Tonifying Shen-Yang and Tonifying Shen- Yin principles have been established to suppress osteoclast- 1. Song YN, Zhang GB, Zhang YY, Su SB. 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