PART 2 Anti-Osteoporotic Activity Of An Edible Traditional Chinese Medicine Cistanche Deserticola On Bone Metabolism Of Ovariectomized Rats Through RANKL/RANK/TRAF6- Mediated Signaling Pathways
Mar 06, 2022
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DISCUSSION
Nowadays, natural extracts and compounds with anti-osteoporotic activity but few side effects have attracted the interest of plenty of pharmaceutical scientists. TCM, for more than a millennium, has been extensively used, apparently safely and effectively, to alleviate various symptoms of diseases including osteoporosis (Lin et al., 2017). It was found TCM that is traditionally used to invigorate and keep kidney essence, where also can be used to treat osteoporosis, including Epimedium brevican Maxim., Eucommia ulmoides Oliver., Cuscuta Chinensis Lam., Dipsacus asper Wall. ex Henry, etc. (Yang et al., 2011; Tao et al., 2017; Qi et al., 2018). Numbers of antiosteoporotic bioactive compounds, including polyphenol and phenylethanoid glycosides, were identified and isolated from dozens of natural medicinal herbs (Ma et al., 2015; Ye et al., 2015; Xu et al., 2017; Liu et al., 2018). C. deserticola is an important natural health food and classic TCM has long been used as a common tonic in China and Japan. It is found to possess a favorable safety profile and broad medicinal functions for the treatment of kidney deficiency, muscle debility, and lumbar weakness. According to the theory of TCM and our previous experiments (Liu et al., 2018), TCM that traditionally used to invigorate kidneys was generally applied to prevent osteoporosis, therefore indicating C. deserticola maybe possess anti-osteoporotic properties. Several published experimental studies had discovered the therapeutic effect of C. deserticola extracts and C. deserticola polysaccharide as well as Cistanche herb on osteoporosis (Liang et al., 2011; Li et al., 2012; Liang et al., 2013; Song et al., 2018), However, the detailed mechanisms of the anti-osteoporotic effect of CDE, especially the upstream signaling,
were remain unclear. Now, in the present study, an OVX rat model which could simulate postmenopausal osteoporosis was used to investigate the anti-osteoporosis action of C. deserticola in vivo, and the proteins levels of RANKL, RANK, and TRAF6 as well as other key regulators related to bone resorption were also analyzed to estimate the possible mechanisms. OVX induced postmenopausal osteoporosis accompanied by a sharp decline in bone quality, bone microarchitecture, uterine, and vagina wet weights, as well as an obvious enhancement in bone resorption and body weight, of which were in part due to estrogen loss (Nian et al., 2009). In the present in vivo experiment, CDE showed potent anti-osteoporosis activity evidenced by the enhanced total BMD and improved trabecular bone microarchitecture, and the increased body weight and decreased uterine and vagina weights of the OVX rats were not affected by CDE treatment, but significantly reversed by EV administration, which implied that CDE could enhance the bone formation without inducing the side effects on the body and uterine organic tissues. BMD is generally used in clinical for the diagnosis of osteoporosis and a low level of BMD is considered as a major risk for bone fractures (Kanis et al., 2015). Besides the BMD, numerous lab experiments and clinical trials revealed that bone microarchitecture, especially the trabecular microarchitecture profile was also significantly contributed to bone quality (Le Corroller et al., 2013), thus making the micro-CT 3D-image evaluation are meaningful in osteoporosis diagnosis nowadays. Our previous study revealed that OVX resulted in a sharp decline in the content of BMC, TMC, Tb. N, and Tb. Th, and increase in the level of Tb. Sp (Ma et al., 2015). In the present research, the bone trabecular microarchitecture structural parameters obtained by analyzing the micro-CT 3D-image of ROI showed CDE was effective in ameliorating trabecular microarchitecture and thus enhanced bone quality. Furthermore, the contents of Ca and P of OVX rats were also employed to reflect the anti-osteoporotic effect. However, totally different from the previously published studies which the urinary excretion levels of Ca and P in OVX rats were significantly increased after the ovariectomized surgery as compared to sham rats (Nian et al., 2009), our data demonstrated that the level of P in the urine of OVX rats was obviously declined (p < 0.001), almost three times less than the rats of a sham. To analyze this diametrically opposed phenomenon, the levels of Ca and P in the serum of OVX rats can not be ignored. It was discovered that although the urinary excretion level of P was significantly decreased in OVX rats, the serum Ca and P levels also showed non-statistically significant declining trends, those both decreased trends maybe ultimately result in a bone loss. Expectedly, CDE significantly increased serum Ca and P levels as well as decreased urinary P excretion induced by ovariectomized surgery, which further proved the fact that CDE could prevent bone loss. Concerning the effect of CDE on bone metabolic index including ALP, BGP, TRAP, DPD, and cathepsin K, similar to the published data that ALP activity in the OVX model group showed a non-statistically increasing trend which indicating an accelerated rate of bone turnover (Swaminathan, 2001) in postmenopausal osteoporosis, CDE treatment showed significant stimulation on ALP activity and also suppression on TRAP, DPD, and cathepsin K properties implied that the therapeutic effect of CDE on OVX rats was mainly through inhibiting bone resorption. Ovariectomized surgery was proved to generally cause oxidative stress and impaired bone antioxidant system in adult rats (Muthusami et al., 2005).
Cistanche deserticola has many effects, click here to know more
In our manuscript, OVX induced a high MDA level in rats. However, the content of MDA in OVX rats was significantly decreased by CDE intervention as compared to the model group (p < 0.001) which indicated the antioxidant activity of this herb. According to the Chinese “yin&yang” and “kidney dominate bone” theory, tonic herbs generally possessed antioxidant activity, and this property always correlated well with the number of phenolic compounds that exist in plants (Ou et al., 2003). And in our present study, an HPLC method was used to discover the phenolic compounds and other chemicals responsible for the anti-osteoporotic activity of CDE. Seven main phenylethanoid glycosides components include echinacoside, cistanoside A, acteoside, isoacteoside, acteoside C, 2’-acetylacteoside, and 6’-acetylacteoside were found in this extract. The structures of the above seven phenylethanoid glycosides were all rich in phenolic hydroxyl groups. Therefore, both the published and our experimental data revealed plenty of phenolic compounds in this plant, all of which supported the antioxidant property of C. deserticola, thus maybe could prevent bone loss by improving the bone antioxidant system. However, the structure-activity relationship between the different phenylethanoid glycosides and their antioxidant properties was still unclear. Only the number of phenolic hydroxyl groups can not totally reflect the anti-osteoporotic activities of phenylethanoid glycosides in C. deserticola, the other factors including the compact, ramification, and symmetric of structures also influenced the effectiveness of the compounds (Chen et al., 2016; Chen et al., 2018a; Chen et al., 2018b; Teng et al., 2018; Chen et al., 2019), thus need us to estimate the related structure-activity relationship in our further studies. As osteoporosis was mainly caused by excessive osteoclastic bone resorption, the factors and regulators related to the activation and differentiation of osteoclast were believed as crucial targets for preventing bone loss. RANKL, a member of the tumor necrosis factor (TNF) family which was expressed in osteoclast precursors, could regulate the differentiation, survival, and activation of osteoclasts when it binds to its receptor RANK, and this process can not be initiated until TRAF6 was recruitment. In our manuscript, the expressions of RANKL, RANK, and TRAF6 were significantly down-regulated and the OPG was up-regulated by CDE treatment, it could be expected that the binding quantities of RANKL with RANK was decreased, together with the limited amounts of TRAF6 could be recruited, thus the differentiation and activation of osteoclasts were suppressed and the bone resorption was inhibited. The binding of RANKL/RANK/TRAF6 triggered a series of downstream signaling cascades including NF-κB and PI3K/AKT pathways. NF-κB was proved essential for osteoclastogenesis as the disruption of NF-κB could lead to an impaired osteoclast differentiation with an osteoporotic phenotype (He et al., 2012), and NF-κB up-regulated c-Fos and down-regulated NFAT2 expressions during RANKL/RANK/ TRAF-induced osteoclastogenesis (Takatsuna et al., 2005). c-Fos regulated expressions of NFAT2 in osteoclasts to mediate its osteoclastogenic effects, and NFAT2 conversely mediated RANKL-induced osteoclast formation, and its overexpression could rescue osteoclastogenesis in c-Fos cells (Boyce et al., 2005). Our data showed that the expressions of IKKβ, NFκBIA, and NFAT2 were down-regulated whereas the level of c-Fos was up-regulated by CDE treatment, indicating that CDE significantly suppressed RANKL/RANK/TRAF-induced NF-κB stimulation, and the activation of c-Fos was promoted and NFAT2 was consequently inhibited. Summarily, the therapeutic effect of CDE on OVX rats main through preventing RANKL/RANK/ TRAF-induced NF-κB activation and PI3K/AKT inactivation as well as c-Fos stimulation and NFAT2 suppression, and finally the differentiation of osteoclast was inhibited and the corresponding bone resorption was suppressed.
ETHICS STATEMENT
For the animal experiment, all procedures were performed in strict accordance with the protocol approved by the institutional animal care and use committee guide of Ningxia Medical University
AUTHOR CONTRIBUTIONS
X-QM and YJ conceived the experiments. BZ, S-QD, and L-LY performed animal experiments. J-JL performed the western blot tests. Y-HD wrote the draft of the manuscript. Y-TL, NL, and X-JZ analyzed the data. C-LH revised the manuscript. All authors reviewed the manuscript.
FUNDING
This work was supported by grants from the National Natural Science Foundation of China (No.81560684); Science Technology Foundation of Higher Education of Ningxia (NGY2017090); Ningxia key research and invention program of science and technology cooperation of the East and the West (No. 2017BY084, 2017BY079); the West Light Foundation of the Chinese Academy of Sciences-Young Scientists of West 2017.
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