Suppression of Inflammation, Osteoclastogenesis and Bone Loss by PZRAS Extract

Panax ginseng has a wide range of activities including a neuroprotective effect, skin protective effects, enhanced DNA repairing, anti-diabetic activity, and protective effects against vascular inflammation. In the present study, we sought to discover the inhibitory effects of a mixture of natural products containing Panax ginseng, Ziziphus jujube, Rubi fructus, Artemisiae asiaticae and Scutellaria baicalensis (PZRAS) on osteoclastogenesis and bone remodeling, as neither the effects of a mixture containing Panax ginseng extract, nor its molecular mechanism on bone inflammation, have been clarified yet. PZRAS upregulated the levels of catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GSH-R) and glutathione peroxidase (GSH-Px) and reduced malondialdehyde (MDA) in LPS-treated RAW264.7 cells. Moreover, treatment with PZRAS decreased the production of IL-1β and TNF-α. PZRAS also inhibited osteoclast differentiation through inhibiting osteoclastspecific genes like MMP-2, 9, cathepsin K, and TRAP in RANKL-treated RAW264.7 cells. Additionally, PZRAS has inhibitory functions on the RANKL-stimulated activation of ERK and JNK, which lead to a decrease in the expression of NFATc1 and c-Fos. In an in vivo study, bone resorption induced by LPS was recovered by treatment with PZRAS in bone volume per tissue volume (BV/TV) compared to control. Furthermore, the ratio of eroded bone surface of femurs was significantly increased in LPStreated mice compared to vehicle group, but this ratio was significantly reversed in PZRAS-treated mice. These results suggest that PZRAS could prevent or treat disorders with abnormal bone loss.


oxidative stress and inflammation processes.
Panax ginseng is a traditional herbal medicine employed as a general health-promoting tonic. Ginsenoside-Rg2 is the main active component that can be extracted from the root and stem leaves of P. ginseng. Additionally, ginsenoside-Rg2 has been known to act through a variety of mechanisms to mediate a neuroprotective effect, a skin protective effect, enhanced DNA repair, anti-diabetic activity and a protective effect against vascular inflammation [22,23]. Ziziphus jujube has been used as herbal medicine and to cure oxidative stress-related diseases such as tumors and cardiovascular diseases [24]. Recently, goshonoside-F5 from Rubi fructus was reported to inhibit the Nuclear factor-κappa B (NF-κB) and MAPK signaling pathways [25]. The extract of Astragalus membranaceus has displayed anti-inflammatory and antioxidant effects in vitro [26]. Some compounds from Artemisia argyi supply a new lead compound for inflammatory disorders [27]. Z. jujube and Scutellaria baicalensis were known to have potential bone-forming effects [28]. However, there has been little investigation into the effect of mixed medicinal plant extracts containing P. ginseng on bone lytic disorders.
Here, we explored the antioxidant, anti-inflammatory, and bone protective effects of mixed extracts of P. ginseng, Z. jujube, R. fructus, A. asiaticae and S. baicalensis (PZRAS) in vitro and in mice.

PZRAS Preparation Process
The plant extracts were prepared, organized and purified by Hanpoong Pham & Foods Co., Ltd. ( Korea). Briefly, P. ginseng was extracted with 60% ethanol while Z. jujube, R. fructus, A. asiaticae, and S. baicalensis were extracted with 30% ethanol, respectively. P. ginseng, Z. jujube, A. asiaticae, and S. baicalensis were obtained from Dong Kyung Pharm Co., Ltd. (Korea) and R. fructus was purchased from Omniherb Co., Ltd. (Korea). Each sample solution was separated through a 5 μm membrane filter, evaporated under reduced pressure and dried at 70-80 o C. For experiment, powdered samples were weighed and mixed at a mass ratio of 4:1:1:1:1 (w/w). DMSO was used to dissolve PZRAS, which was then diluted in medium for cell culture as needed.

Lipid Peroxidation (MDA) and Enzyme Activity Assays
Thiobarbituric acid (TBA) was used to determine the amount of lipid peroxidation as previously identified [30]. The absorbance of solution was read at 532 nm and calculated based on the ε value of 153,000.
Catalase (CAT) activity was measured by Aeby's method [31]. Superoxide dismutase (SOD) and GSH-Px activity were determined using an SOD determination kit (Oxis Research, USA) and a commercially available kit (Sigma-Aldrich) according to the manufacturers' guidance, respectively.

Western Blot Analysis
Western blotting was carried out by previously reported method [32]. In brief, whole cell lysates were obtained using radioimmuno-precipitatiotion assay (RIPA) buffer (iNtRON Biotechnology, Korea) containing 1 mM phenylmethylsulfonylfluoride (PMSF), and 1× protease inhibitor cocktail. Proteins were separated using SDS-PAGE and transferred subsequently onto PVDF membrane (Bio-Rad, USA) through a wet transfer system at 100 V and 350 mA. The protein bands were detected using chemiluminescence (ECL Plus Kit, Amersham Biosciences, USA) and β-actin typically was utilized as a loading control.

Enzyme-Linked Immunosorbent Assay (ELISA)
To assess the secreted levels of pro-inflammatory cytokine (TNF-α and IL-1β), cells were pre-treated with PZRAS for 30 min and stimulated by LPS (2 μg/ml) for 8 h and 24 h, respectively. TNF-α and IL-1β ELISA kits (R&D Systems, USA) were used to measure concentrations of cytokines in culture supernatants.

Lipopolysaccharides (LPS)-Induced Bone Resorption
Animal experiments were performed under the guidance of the Jeonbuk National University Laboratory Animal Center (Approval no. CBNU 2018-094, Korea). Previous studies revealed that LPS leads to inflammatory bone resorption by direct intraperitoneal injection in mice as a bone erosion animal model [33]. Briefly, six-weekold ICR mice were injected intraperitoneally on days 1, 4, 7, 11 with or without LPS (5 mg/kg body weight). PZRAS (100, 200 μg/kg and 400 μg/kg of body weight) was administered orally 1 day prior to LPS injection and every day thereafter for 14 days. Six week-old male ICR mice used during the experiments were obtained from Damool Science (Korea).

Microcomputed Tomographic Bone Analysis
The femurs on the left sides of the mice were scanned with a 1076 Micro CT System (Skyscan, Belgium), as previously described [34]. A three-dimensional image reconstruction analysis system was used to assess bone volume / tissue volume fraction (BV/TV, %).

Histological Bone Analysis
Paraffin section and staining preparation were determined as described previously [32]. The femurs of the other side were processed through fixation, decalcification, embedding, sectioning, and H&E staining. Images were then obtained by a color digital video camera (ZEISS, West Germany) at 25 × and 100 × magnifications.

Statistical Analysis
All the experiments were repeated at least three times and expressed as means ± S.D., unless otherwise indicated. Statistical analyses were performed using SPSS ver. 12.0 software, and P-values less than 0.05 were considered statistically significant.

Effect of PZRAS on DPPH Radical Scavenging Activity
DPPH radical scavenging analysis was extensively used to measure the antioxidant effect of biological samples [35]. The radical scavenging activity of PZRAS was compared with vitamin C as the standard antioxidant. As shown in Table 1, PZRAS exhibited radical scavenging activity with an IC 50 of 16.0 ± 1.4 μg/ml, whereas vitamin C showed 10.4 ± 2.1 μg/ml. Therefore, PZRAS has a radical scavenging activity comparable to vitamin C.

Effects of PZRAS on Antioxidant Enzymes
To confirm whether PZRAS exhibited an antioxidant activity, activities of CAT, SOD, GSH-R, and GSH-Px and MDA level were measured. As shown in Table 2, CAT activity was significantly enhanced by a 10 μg/ml concentration of PZRAS (4.43 ± 0.05 units/mg) as well as in a 50 μg/ml (4.46 ± 0.04 units/mg) concentration compared with LPS-treated group (3.90 ± 0.03 units/mg). Moreover, the activity of SOD significantly increased in 10 and 50 μg/ml PZRAS (98.0 ± 3.7 and 108.1 ± 2.8 units/mg) concentrations compared with the LPS-alone group (61.6 ± 4.8 units/mg). PZRAS treatment markedly suppressed GSH-Px and GSH-R in LPS-induced RAW264.7 cells. However, GSH-Px and GHS-R activity significantly increased in concentration of 1 μg/ml PZRAS (33.9 ± 0.92 and 566.1 ± 13.90 units/mg, respectively). The MDA level was higher than control in LPS-stimulated RAW264.7 cells, whereas PZRAS markedly reduced MDA level in 10 and 50 μg/ml concentrations compared with LPS-only group (Fig. 2). The results suggest that high doses (10 and 50 μg/ml) of PZRAS are more potent than low doses and PZRAS exhibits effective antioxidant capacity in LPS-treated RAW 264.7 cells.

Effect of PZRAS on LPS-Induced Pro-Inflammatory Cytokines
In order to explore the anti-inflammatory effect of PZRAS, RAW264.7 cells were treated with or without PZRAS. As shown in Fig. 3A, the stimulation with LPS increased the iNOS protein expression in RAW264.7 cells. However, 50 μg/ml of PZRAS downregulated LPS-induced iNOS protein level compared with LPS-only group (p < 0.05).
Generally, TNF-α and IL-β are released by LPS-stimulated macrophage cells and are associated with increased inflammatory responses [36]. To better perceive the effect of PZRAS on inflammation, secreted levels of TNF-α and IL-β in the cultured medium of RAW 264.7 cells were measured by ELISA. TNF-α level was markedly upregulated by LPS treatment, but PZRAS at 50 μg/ml concentration significantly decreased this induction (p < 0.05) (Fig. 3B). The level of IL-1β was increased by LPS treatments, and this increased level of IL-1β was strongly suppressed by PZRAS (Fig. 3C). These results indicated that PZRAS can manipulate the LPS-induced proinflammatory activity of cytokine, TNF-α and IL-β.  October 2020 ⎪ Vol. 30 ⎪ No. 10

Effects of PZRAS on RANKL-Induced MAPK Expression in RAW264.7 Cells
The mitogen-activated protein kinases, ERK, JNK, and p38 were stimulated through RANKL-RANK signaling pathway in osteoclast precursor cells or RAW264.7 cells [39,40]. To clarify the intracellular mechanism of PZRAS in RANKL-signaling pathway, activities of ERK, JNK, and p38 were examined by western blot analysis. As shown in Fig. 5, RANKL at 50 ng/ml activated all three MAPKs in RAW264.7 cells, whereas PZRAS downregulated the activities of ERK and JNK but not p38 (Fig. 5).

Effects of PZRAS on NFATc1 and c-Fos in RAW264.7 Cells
The RANKL-RANK axis resulted in the activation of RANKL-induced transcription factors such as NFATc1 or c-Fos during osteoclast formation [41,42]. In a next step, effects of PZRAS on the expressions of NFATc1 or c-Fos were investigated during osteocalstogenesis of RAW264.7 cells. RANKL upregulated mRNA expressions of NFATc1 or c-Fos compared with control group but 50 μg/ml of PZRAS significantly reduced those transcription activities (Fig. 6A). RANKL also increased the protein expressions of NFATc1 and c-Fos within 12 h in RAW264.7 cells. However, PZRAS inhibited those protein levels compared to untreated group (Fig. 6B). These results imply that PZRAS can prevent osteoclast maturation by inhibiting RANKL-induced NFATc1 and c-Fos.

Effect of PZRAS on LPS-Treated Bone Lysis
To examine the suppressive effect of PZRAS on bone lysis, 6-week-old mice were injected with LPS without or with different PZRAS. We also examined the efficiency of PZRAS for bone loss by comparing with an antioxidant complex (85 mg/kg vitamin D and 1 μg/ml Ca 2+ ). Micro-CT images revealed that LPS induced serious bone destruction in murine femurs. In contrast, PZRAS significantly diminished LPS-induced bone destruction at the indicated concentrations (Fig. 7A). The microstructural indices in femurs revealed that LPS injection significantly decreased bone volume/tissue volume (BV/TV) compared with vehicle group (p < 0.05). However, those reductions were markedly reversed by PZRAS at 200 or 400 mg/kg (Fig. 7B). H&E stain revealed that osteoclast maturation and bone loss by LPS were significantly recovered by treatment with PZRAS. Although LPS significantly caused bone loss in the femurs, PZRAS protected them from bone loss (Fig. 7C). Although the parameter of eroded bone surface per bone surface (ES/BS) of femurs significantly increased in the LPS-injected     mice compared to vehicle group (p < 0.05), this ratio was significantly recovered in PZRAS-treated mice (p < 0.05) (Fig. 7D). Taken together, the above results strongly suggest that PZRAS can inhibit osteoclast differentiation and bone loss.

Discussion
Natural flavonoid compounds are good targets of therapeutic agents because they are not associated with severe risk and possibility of long-term treatment. Therefore, the selection of natural compounds can be important to develop therapeutics for osteoporosis [7].
In this study, various compounds of flavonoids, P. ginseng, Z. jujube, Rubi fructus, A. asiaticae and S. baicalensis (PZRAS) were selected for anti-inflammatory activity and examined whether they had a protective effect against bone loss. Thus, we elucidated the anti-inflammatory activities of PZRAS in both in vitro and in vivo systems. The dual activities against iNOS and inflammation cytokine IL-1β were associated with increased antioxidant properties (CAT, SOD, GSH-Px and GSH-R). In addition, PZRAS has shown significant efficacy in protecting from LPS-induced bone loss, suggesting its potential therapeutic usage for bone disorders.
In previous studies, LPS can activate the productions of TNF-α, IL-1β, NO and other oxidative parameters [43,44]. Moreover, abnormal expressions of TNF-α, IL-1β, and NO are associated with chronic inflammatory diseases [45]. Therefore, the suppression of pro-inflammatory cytokines and regulators could be applied for treating immune disorders [36]. A recent study demonstrated that P. ginseng leaf extract including ginsenosides Rd and Km inhibited TNF-α-enhanced expression of iNOS in HepG2 cells [46]. In this study, PZRAS dramatically repressed the expression of iNOS as well as secretion of IL-1β and TNF-α in LPS-stimulated RAW264.7 cells. These results suggest that PZRAS may exhibit the anti-inflammatory capacity through suppression of proinflammatory factors such as iNOS, IL-1β or TNF-α.
Free radical attack on plasma membrane components such as LPS generates MDA. GSH is a strong antioxidant for cellular detoxification processes which can reduce MDA production. Therefore, it has been suggested that endogenous GSH plays an essential role in preventing oxidative stress-related inflammation [13]. In this study, PZRAS effectively increased activities of CAT, SOD, and GSH-Px and markedly decreased the MDA level (Table 2 and Fig. 2). These results suggest that PZRAS may act as an antioxidant agent via upregulation of CAT, SOD, and GSH-Px activities and suppression of MDA production.
Previous studies have demonstrated that the RANK/RANKL axis leads to activation of several downstream signaling pathways for osteoclast-specific genes and transcription factors [38][39][40]. For instance, MAPKs regulate NFATc1 and c-Fos, transcription factors which then modulate the MMP-9 mRNA expression during osteoclast maturation [41,42]. PZRAS significantly inhibited phosphorylated ERK and JNK, (but not p38 expression), mRNA expressions of MMP-2, -9, TRAP, cathepsin K as well as transcription factors (NFATc1, c-Fos) during osteoclast differentiation. We also demonstrated that PZRAS efficiently worked to cure bone loss in LPS-induced animal model as evidenced by its improving BV/TV and ES/BS.
The above results clearly show that PZRAS exhibits an anti-osteoclastogenic potential by reducing the generation of iNOS, IL-1β, and MMP-2, -9, and also increased levels of antioxidant enzymes (CAT, SOD, GSH-Px and GSH reductase). PZRAS also prevented LPS-induced bone destruction in mice. Therefore, our findings suggest that PZRAS could be a promising therapeutic candidate for various bone-related diseases.