Cissus quadrangularis (Hadjod) Inhibits RANKL-Induced Osteoclastogenesis and Augments Bone Health in an Estrogen-Deficient Preclinical Model of Osteoporosis Via Modulating the Host Osteoimmune System

Osteoporosis is a systemic skeletal disease characterised by low bone mineral density (BMD), degeneration of bone micro-architecture, and impaired bone strength. Cissus quadrangularis (CQ), popularly known as Hadjod (bone setter) in Hindi, is a traditional medicinal herb exhibiting osteoprotective potential in various bone diseases, especially osteoporosis and fractures. However, the cellular mechanisms underpinning its direct effect on bone health through altering the host immune system have never been elucidated. In the present study, we interrogated the osteoprotective and immunoporotic (the osteoprotective potential of CQ via modulating the host immune system) potential of CQ in preventing inflammatory bone loss under oestrogen-deficient conditions. The current study outlines the CQ’s osteoprotective potential under both ex vivo and in vivo (ovariectomized) conditions. Our ex vivo data demonstrated that, in a dose-dependent manner CQ, suppresses the RANKL-induced osteoclastogenesis (p < 0.001) as well as inhibiting the osteoclast functional activity (p < 0.001) in mouse bone marrow cells (BMCs). Our in vivo µ-CT and flow cytometry data further showed that CQ administration improves bone health and preserves bone micro-architecture by markedly raising the proportion of anti-osteoclastogenic immune cells, such as Th1 (p < 0.05), Th2 (p < 0.05), Tregs (p < 0.05), and Bregs (p < 0.01), while concurrently lowering the osteoclastogenic Th17 cells in bone marrow, mesenteric lymph nodes, Peyer’s patches, and spleen in comparison to the control group. Serum cytokine analysis further supported the osteoprotective and immunoporotic potential of CQ, showing a significant increase in the levels of anti-osteoclastogenic cytokines (p < 0.05) (IFN-γ, IL-4, and IL-10) and a concurrent decrease in the levels of osteoclastogenic cytokines (p < 0.05) (TNF-α, IL-6, and IL-17). In conclusion, our data for the first time delineates the novel cellular and immunological mechanism of the osteoprotective potential of CQ under postmenopausal osteoporotic conditions.


Introduction
Osteoporosis is a progressive, systemic skeletal disease characterised by low BMD, degeneration of bone micro-architecture, and impaired bone strength which makes the bone prone to developing fragility-related fractures. Among the elderly population, osteoporosis is particularly prevalent in postmenopausal women and patients undergoing long-term mechanism by which CQ inhibits inflammatory bone loss in estrogen-deficient settings. Based on this evidence, we examined the osteoprotective and immunoporotic potential of CQ. Altogether, results from the present study provide compelling evidence in favor of the anti-osteoclastogenic and immunoporotic (the osteoprotective potential of CQ via modulating the host immune system) potential of CQ in ameliorating inflammatory bone loss in a preclinical model of PMO.

Animals and Treatments
For our in vivo experiments, female Balb/c mice, age (8-10 weeks), and weight (23 ± 2 gm) were procured and housed in polystyrene cages at the institutional animal facility (Department of Pharmaceutical Sciences, Dr. Harisingh Gour Vishwavidyalaya, Sagar, Madhya Pradesh, India), under a specific pathogen-free (SPF) atmosphere with constant humidity (45-65%), temperature, and a 12-h light-dark cycle. Experimental mice had unlimited access to a conventional mouse diet and water. Mice were acclimatized for one week in laboratory conditions before ovariectomy. Post-acclimatization mice underwent ventral bilateral ovariectomy (Ovx), after anesthetizing intraperitoneally with Ketamine (100-150 mg/kg) and Xylazine (5-16 mg/kg) according to the standardized protocols and procedures. One-week post-surgery, mice were randomly divided into three groups, i.e., Sham-operated, Ovx, and Ovx + CQ with six mice/group.
The numbers of animals used in our experiments were based on statistical power calculations with parameters from our extensive previous bone health-related experiments in our lab. Bone Mineral Density (BMD) was used as the parameter for power calculations. From our previous study , we found that the BMD of the bone of ovx is 1.74 gm HA/cm 3 (mean) and the BMD of the bone of sham mice is 2.8 gm HA/cm 3 (mean). Standard Deviation (SD) for this experiment was 0.65 gm HA/cm 3 . From this data, we selected the level of significance at 5% and the power of study at 80%. The effect size (mean of two groups) in this situation is 1.03. Hence, the sample size was calculated as follows: sample size = 2SD 2 (Z score value of level of significance (5%) + (Z score value of power of the study (80%) / effect size = 2 x 0.4225 (1.96 +0.842) 2 /(1.03) 2 = 6.44. Thus, we arrived at using six mice per group in our current study.
The Ovx + CQ group mice were orally administered with Cissus quadrangularis crude extract at a dose of 500 mg/kg/day using mice oral feeding needles, twice daily for 45 days. On the final day of the experiment, mice were euthanized via CO 2 asphyxiation, followed by the collection of bone, lymphoid tissues, and blood samples through retro-orbital sinus for further analysis. All the investigational procedures were followed according to the prior permission available with the submitted protocols to The Committee for Control and Supervision of Experiments on Animals (CPCSEA), India with former sanction from Institutional Animal Ethical Committee (IAEC), Dr. Harisingh Gour Vishwavidyalaya, Sagar, Madhya Pradesh, India, with CPCSEA, Registration No. (379/CPCSEA/IAEC-2018/017).

Collection of Plant Material
Excellent quality CQ stems were collected from the Botanical Garden, Dr. Harisingh Gour Vishwavidyalaya, (A Central University), Sagar, (M.P.), India, and authenticated at the Department of Botany, Dr. Harisingh Gour Vishwavidyalaya, Sagar, (M.P.), India. The voucher specimen no. BOT/H/02/50/04 has been kept in the herbarium file of the same department.

Preparation of CQ Extract
CQ stems weighing 1.5 kg were collected, washed (tap water), air-dried, and ground into a coarse powder. The powdered stems were then extracted with 90% (v/v) ethanol by using the Soxhlet apparatus at 60 • C [32,33]. The resultant CQ crude extract was filtered, and the extra solvent was removed by a vacuum evaporator for experiments. The yield was calculated (150 g) with a syrup-like consistency and the extract was stored at 4 • C for further investigations.

Osteoclasts Differentiation and TRAP Staining
Primary bone marrow-derived macrophages (BMMs) were obtained from the mouse bone marrow cavity by flushing both tibia and femur dissected out of the 8-10-week-old, female Balb/c mice, as reported previously [16,34]. Briefly, the bone marrow cells were further suspended in complete α-MEM media containing 10% heat-inactivated fetal bovine serum (FBS). Subsequently, red blood cells (RBCs) were lysed by using 1X RBC lysis buffer, followed by overnight culturing of cells in complete α-MEM media supplemented with MCSF (35 ng/mL). The non-adherent cells were collected and the following day and seeded in an osteoclastogenic medium with RANKL (100 ng/mL) and MCSF (30 ng/mL) with or without CQ extracts at different concentrations, i.e., 10, 20, 50, and 100 g/mL for f consecutive days. On the third day, the culture media was replenished by replacing half of the media with media containing fresh osteoclastogenic factors. To determine the presence of mature multinucleated osteoclasts (with >03 nuclei), cells were then treated with a fixative solution containing formaldehyde, acetone, and citrate for 10 min at 37 • C. Following fixation, cells were stained with TRAP according to the manufacturer's instructions. Osteoclasts with a nucleus count of ≥ 3 were regarded as mature osteoclasts. An inverted microscope (ECLIPSE, TS100, Nikon, Japan) was used to count and capture images of multinucleated TRAP + cells. Image-J (NIH, Bethesda, MD, USA) software was used to count and measure the area of TRAP + cells.

F-Actin Ring Polymerization Assay
The F-actin ring polymerization experiment was performed as previously described [34]. Specifically, bone marrow-derived monocytes/macrophages (osteoclast precursors) were seeded on glass coverslips in 12-well plates with or without CQ at two distinct concentrations, i.e., 50 and 100 µg/mL. On day 4, cells were rinsed twice with 1X PBS and fixed with 4% paraformaldehyde (PFA) for 20 min. The cells were then treated with 3% BSA for 30 min to prevent non-specific binding, and then stained with FITC-labelled-phalloidin for an hour at room temperature in the dark. Finally, cells were stained for 5 min in the dark with DAPI (10 µg/mL), and the slides were further examined and imaged using an immunofluorescence microscope (Imager Z2, Zeiss).

Cell Viability or Metabolic Activity Assay
To determine the viability of the cells in relation to their metabolic activity, the MTT test was used. Briefly, BMMs were seeded in 96-well plates at a density of 10,000 cells per well and cultured for 24 h at 37 • C in a CO 2 incubator. Cells were exposed to CQ at various doses the next day and were further incubated for 48 h. Following the completion of the treatment, MTT (0.5 mg/mL) was added, and the plate was incubated in a CO 2 incubator at 37 • C for 4 h. After incubation, DMSO was used to dissolve the formazan crystals. The plate was orbitally shaken for 5 s, and a reading at 570 nm was obtained using a microplate reader (Synergy H1, BioTek)

Scanning Electron Microscopy (SEM)
SEM images of the cortical regions of the femur bones from distinct groups were collected in accordance with the previous description [14,15]. Briefly, the femur cortical bones dissected were submerged in triton X-100 (1%), for 2-3 days. After the completion of 3 days, the bone samples were again resuspended in 1X PBS for further investigations. Bone slices were cut and stored under an incandescent lamp for one day before to analysis to dry the bone samples. The dried bone samples were then sputter-coated with platinum. Next, the platinum-coated samples were analyzed by a Leo 435-VP SEM outfitted with a 35 mm camera and digital imaging. The final step was to collect SEM images of the targeted cortical regions from the comparable regions of the various groups at three different magnifications i.e., 10X (lower) 1000X (intermediate), and 10,000X (higher). MATLAB software was further used to analyze the captured images (Math Works, Natick, MA, USA).

Micro-Computed Tomography (µ-CT)
To analyze the trabecular areas of the femur, tibia, and lumbar vertebrae-5 (LV-5) as previously described, µ-CT was carried out using a Sky-Scan 1076 scanner (Aartselaar, Belgium) [14,34]. After loading the samples, a 0.5 mm aluminum filter was applied, the exposure time was adjusted to 590 ms, and scanning was done at 50 Kv, 201 mA, with each sample oriented precisely for analysis. With a resolution of 9 m/pixel, 1800 pictures in total were collected. The reconstruction of the desired bone samples was then finished using NRecon software. Next, for performing the segregation of growth plates from the adjoining tissues, manual segmentation was carried out to obtain 2D segments of sagittal radiographs. It was then preceded by the reconstruction step for gaining the 3D µ-CT images. Next, the images obtained were analyzed for choosing the growth plate's true height with the help of the data viewer tool. Region of interest (ROI) was drawn in secondary spongiosa for a total of 100 slices to analyze the trabecular bones. The primary spongiosa and cortical portions of the bone samples were excluded, and it was positioned 1.5 mm from the distal end of the growth plates. Similar to this, a total of 50 uninterrupted slices were included for the in vivo measurements of the LV-5 trabeculae, starting from the beginning of the trabecular region of the bone within the vertebral body [35,36]. To further examine the cortical bone microarchitectural details, 350 subsequent image slices were discarded from the trabecular area of the bone behind the growth plates. Finally, only 200 consecutive image slices out of these 350 slices were chosen for further analysis utilizing the CTAn software in vivo. Various 3D-histomorphometric trabecular analyses such as BV/TV (bone volume/tissue volume), Tb.Sp (trabecular separation), Tb.Th (trabecular thickness), and 2D cortical parameters like Tt.Ar (total cross-sectional area inside the periosteal envelope), Ct.Ar/Tt.Ar (cortical area fraction), and Ct.Th (cortical thickness) was considered [18,35,37]. The BMD of the femur, tibia, and LV-5 regions of the bones were then estimated using the volume of interest (VOI) gathered for µ-CT scans made from both cortical and trabecular portions of the bones. The results were obtained by calibrating using hydroxyapatite phantom rods with a 4 mm diameter and known BMD values: 0.25/cm 3 and 0.75/cm 3 [14,15]. As a result, the estimated BMD was determined for each examination of the desired bone samples using the linear relationship between BMD and µ-CT attenuation coefficient.

Flow Cytometry
Immediately following mice sacrifice, the lymphoid tissues such as BM, SP, MLN, and PP were harvested and homogenized to make a single cell solution. Then, RBCs were lysed with 1X RBC lysis buffer and washed twice with 1X PBS. Further cells were processed for immune cell staining. For analysis of intracellular cytokines, we followed the following protocol: cells obtained from different lymphoid organs were resuspended in RPMI-1640 complete medium containing 10% FBS and seeded in 96 well plates at a density of 1 × 10 6 cells/mL. Cells were further stimulated with a mixture of Ionomycin (1 ug/mL), and PMA (50 ng/mL, Sigma Aldrich). Before harvesting, cells were treated with protein transport inhibitors (BD, USA) containing brefeldin and monensin for 5 h. After harvesting, stimulated cells were stained for both surface and intracellular markers.

Enzyme-Linked Immunosorbent Assay (ELISA)
The levels of IL-4, IL-6, IL-10, IL-17, IFN-γ, and TNF-α proteins were determined from the collected blood serum samples by using commercially available kits (OptEIA BD, Franklin Lakes, NJ, USA) and adhering to the manufacturer's instruction and safety precautions. Multimode microplate reader BioTek Synergy H1, BioTek Instruments (USA) was used for readings as per the requirements.

Histologic Analysis
Femoral bones were decalcified for 15 days via the use of 10 % tetrasodium EDTA aqueous solution by placing it on a rocker. Paraffin-embedded sections (5 µm) from each femur were processed for the histological hematoxylin and eosin staining analysis. Sections were imaged using a microscope.

Statistical Analysis of Data
For performing the statistical analysis of the available data, results were represented as mean ± SEM for at least two independent studies with 6 mice per group. Comparisons were made between three groups, Sham, Ovx, and Ovx + CQ, using one-way analysis of variance (ANOVA), followed by an unpaired student t-test. In comparison to the Ovx group, the statistical significance level of the data was established as p ≤ 0.05 (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

CQ Inhibits RANKL-Induced Osteoclastogenesis
Firstly, we were interested in investigating the anti-osteoclastogenic potential of CQ under ex vivo conditions. This was achieved by performing RANKL-induced osteoclast development in bone marrow cells (BMCs) using osteoclastogenic medium supplemented with M-CSF and RANKL in the presence or absence of CQ at varied doses, i.e., 10, 20, 50, and 100 µg/mL. The multinucleated mature osteoclasts were identified by TRAP-staining after four days of incubation ( Figure 1A). It was observed that CQ treatment greatly reduced the quantity of large multinucleated osteoclasts ( Figure 1B). Moreover, the total number of TRAP + osteoclasts were also significantly reduced (p ≤ 0.001) in a dose-dependent manner relative to the control group ( Figure 1C,D). Additionally, in comparison to the control group, the area of TRAP + multinucleated mature osteoclasts were also markedly diminished in the CQ treatment group ( Figure 1E). Further, we employed MTT assay to rule out the hypothesis that the observed decrease in osteoclast differentiation and number is due to CQ-cytotoxicity, and we observed that CQ treatment at different concentrations has no effect on cell viability ( Figure S1). These findings clearly suggest that CQ has potent anti-osteoclastogenic properties. Values are expressed as mean ± SEM, and equivalent outcomes were observed in two independent experiments. For the indicated groups, p ≤ 0.05 *** p ≤ 0.001) was used as the cutoff for statistical significance. In the bar graphs, red is indicating the positive control and green is indicating the treatment groups. Values are expressed as mean ± SEM, and equivalent outcomes were observed in two independent experiments. For the indicated groups, p ≤ 0.05 *** p ≤ 0.001) was used as the cutoff for statistical significance. In the bar graphs, red is indicating the positive control and green is indicating the treatment groups.

CQ Attenuates the Functional Activity of Osteoclasts
A crucial aspect of mature and functional multinucleated osteoclasts for bone resorption is the formation of F-actin rings. Thus, using an F-actin assay, we next evaluated CQ's impact on osteoclast's functional ability. We observed that in comparison to the control group, CQ treatment considerably decreased both the number as well as the area of F-actin rings in a dose-dependent manner (Figure 2A-D). These findings further supported our above results that CQ not only suppressed the formation of osteoclasts but also robustly inhibited the functional activity of osteoclasts.

CQ Attenuates the Functional Activity of Osteoclasts
A crucial aspect of mature and functional multinucleated osteoclasts for bone resorption is the formation of F-actin rings. Thus, using an F-actin assay, we next evaluated CQ's impact on osteoclast's functional ability. We observed that in comparison to the control group, CQ treatment considerably decreased both the number as well as the area of Factin rings in a dose-dependent manner (Figure 2A-D). These findings further supported our above results that CQ not only suppressed the formation of osteoclasts but also robustly inhibited the functional activity of osteoclasts.  ANOVA was used to analyse the results, and then unpaired Student t-tests were used to compare the indicated groups. Values are expressed as mean ± SEM, and equivalent outcomes were observed in two independent experiments. For the indicated groups, p 0.05 *** p ≤ 0.001) was used as the cutoff for statistical significance. In the bar graphs, red is indicating the positive control and green is indicating the treatment groups.

CQ Augments Bone Health under Postmenopausal Osteoporotic Conditions
Moving ahead we next evaluated the potential of CQ in reducing the inflammatory bone loss under estrogen-deficient conditions in a preclinical model of osteoporosis, i.e., Ovx. For achieving the same, 8-10 weeks old Balb/c (female mice) were arbitrarily divided into three groups, i.e., Sham, Ovx, and Ovx + CQ group. CQ was given orally to Ovx + CQ group mice for 45 days at a dose of 500 mg/kg/day. At the end of the experiment, all the animals were euthanized, and bone samples were harvested for analysis of various bone parameters ( Figure 3A). Firstly, we used SEM to assess CQ's osteoprotective potential. In comparison to the sham group, SEM image analysis of the femur cortical areas revealed the development of larger Howship's lacunae/resorption pits, indicating increased osteoclastogenesis in the Ovx group ( Figure 3B). Surprisingly, CQ administration in the Ovx group resulted in a large reduction in the Howship's lacunae and resorption area, thereby indicating reduced osteoclastogenesis ( Figure 3B). We used MATLAB (matrix laboratory) analysis to determine the degree of homogeneity in 2D-SEM images to increase our understanding of the relationship between bone loss and bone development. In MATLAB analysis, the blue color denotes a lower correlation or lower bone mass. Contrarily, the red color denotes a higher correlation, or larger bone mass. A thorough examination showed that the Ovx + CQ group had more bone mass than the Ovx group, indicating a higher correlation ( Figure 3C). Overall, our SEM data further validates our ex vivo findings that CQ treatment markedly decreased bone resorption in Ovx mice.

CQ Enhances Bone Micro-Architecture and Histomorphometric Indices
To further analyze the potential of CQ in maintaining bone health, we next performed bone micro-architecture and histomorphometric analysis via high-resolution µ-CT, (a gold standard), for calculating various bone morphometric indices. Since, the LV-5 region is crucial in the early detection of osteoporosis or bone loss [15,19], thus we next investigated the effect of CQ in the trabeculae region of LV-5. We observed that the administration of CQ to the Ovx mice group significantly enhanced the bone microarchitecture of the LV-5 trabecular region as compared to the Ovx group ( Figure 4A). In addition, the LV-5 trabecular histomorphometric analysis for various parameters such as BV/TV, Tb.Th, Tb.N, Conn.D, Tb.Sp, and Tb.Pf was also considered. Excitingly, it was observed that BV/TV (p ≤ 0.01), Tb.Th (p ≤ 0.05), Tb.N (p ≤ 0.05), and Conn.D (p ≤ 0.05) parameters were significantly enhanced in the Ovx + CQ group along with a significant decrease in the Tb.Sp (p ≤ 0.05) and Tb.Pf (p ≤ 0.05) ( Table 1). Next, we monitored the histomorphometric parameters of the trabecular and cortical regions of the femoral and tibial bones, where we observed a significant improvement in the 3D micro-architecture of both the trabecular and cortical regions of the CQ administered Ovx group (Figure 4C, E, G, I). Similar results were also observed in trabecular bone indices, i.e., BV/TV (p ≤ 0.01), Tb.Th (p ≤ 0.05), Tb.N (p ≤ 0.05), and Conn.D (p ≤ 0.05) parameters, along with a significant decrease in the Tb.Sp (p ≤ 0.05) and Tb.Pf (p ≤ 0.05) for femur trabecular regions (Table 1). Likewise, we also observed a significant increase in the histomorphometric parameters of the tibia trabecular region along with an improved 3D bone micro-architecture (Table 1). Notably, administration of CQ significantly improved the bone microarchitecture and histomorphometric indices of the cortical region in both femoral as well as tibial bones relative to the Ovx group (Table 1). These results were further corroborated by decalcified H a E-stained bone sections, wherein femoral sections from the Ovx mice showed a dearth of cancellous and trabecular bone regions and, treatment with CQ significantly reverses the same in Ovx mice ( Figure S2). Collectively, our data indicate that CQ treatment dramatically improves both histomorphometric and bone micro-architecture indices and improves bone health in Ovx mice under estrogen-deficient settings.

CQ Enhances Bone Micro-Architecture and Histomorphometric Indices
To further analyze the potential of CQ in maintaining bone health, we next performed bone micro-architecture and histomorphometric analysis via high-resolution µ-CT, (a gold standard), for calculating various bone morphometric indices. Since, the LV-5 region is crucial in the early detection of osteoporosis or bone loss [15,19], thus we next investigated the effect of CQ in the trabeculae region of LV-5. We observed that the   Th; trabecular thickness, Tb.Sp; trabecular separation, Tt.Ar; total cross-sectional area inside the periosteal envelope, Ps.Pm; periosteal perimeter, Ct.Th; average cortical thickness and BMD; bone mineral density. ANOVA was used to analyse the results, and then unpaired Student t-tests were used to compare the indicated groups. Values are expressed as mean ± SEM, and equivalent outcomes were observed in two independent experiments. For the indicated groups, p 0.05 (* p ≤ 0.05, ** p ≤ 0.01) was used as the cutoff for statistical significance. In the figure, black color is indicating control group, purple color is indicating osteoporotic group and green color is highlighting osteoporotic group treated with CQ.  4 ) 0.14 ± 0.02 0.13 ± 0.01 0.31 ± 0.03 * Table 1 Bone histomorphometric indices Trabecular (LV-5, femur, and tibia) and cortical (femur and tibia) bones were studied using bone histomorphometry. Histomorphometric measurements of the LV-5, femur, and tibia in the Sham, Ovx, and Ovx + CQ groups. BV/TV, none volume/tissue volume ratio; Tb.Th, trabecular thickness; Tb.Sp, trabecular separation; Conn.D, connectivity density; Tb.N, trabecular number; Tb.Pf, trabecular pattern factor; Tt.Ar, total cross-sectional area; T.Pm, total cross-sectional perimeter; Ct.Ar, cortical bone area; B.Pm, bone perimeter; Ct.Th, average cortical thickness and J, polar moment of inertia (MMI). ANOVA was used to analyse the results, and then unpaired Student t-tests were used to compare the indicated groups. Values are expressed as mean ± SEM, and equivalent outcomes were observed in two independent experiments. For the indicated groups, p 0.05 (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001) was used as the cutoff for statistical significance.

CQ Improves Bone Mineral Density (BMD)
Since BMD is regarded as a determinant of osteoporotic fractures, we therefore next assessed the BMDs of both the axial and appendicular sites that bear body weight. µCT permits the measurement of BMDs, and our findings clearly demonstrated significant enhancement in the BMDs of the LV-5, femur, and tibia trabecular as well as the cortical regions of the bones, indicating that administration of CQ significantly improves the mineral content of bones ( Figure 4B,D,F,H,J). Altogether, both our µ-CT analysis and BMD data clearly suggest that CQ administration improves BMDs and thus enhances bone health in Ovx mice.

CQ Augments Bone Health Via Modulating Immunoporotic Cells
Recent studies from our group have demonstrated the crucial role of immune systemin the pathophysiology of osteoporosis, which is now referred to as "Immunoporosis". Nu-merous studies along with our own had established the anti-osteoclastogenic role of Th1, Th2, Tregs, and Bregs along with the osteoclastogenic role of Th17 in osteoporosis. Consequently, we next investigated the immuno-modulatory capability of CQ in enhancing bone health. We observed that CQ administration drastically increased the population of antiosteoclastogenic Th1 (CD4 + IFN-γ + ) ( Figure 5A-H), Th2 (CD4 + IL-4 + ) ( Figure 6A-H), Tregs (CD4 + Foxp3 + ) ( Figure 7A-H), and Bregs (CD19 + CD1d hi CD5 + ) ( Figure 8A-H; Figure S4) in various lymphoid organs such as BM (prime site of osteoclastogenesis), SP, MLN, and PP. We further evaluated the percentage of osteoclastogenic Th17 cells (CD4 + Rorγt + ) via flow cytometry in all the groups. Intriguingly, we found that oral treatment of CQ significantly reduced the amount of inflammatory Th17 cells in several lymphoid organs, including the BM, SP, MLN, and PP ( Figure 9A-H). Altogether, our data convincingly demonstrate that oral administration of CQ improves bone health via regulating immunoporotic cells.

CQ Skews the Cytokine Balance under Estrogen-Deficient Conditions
Osteoclastogenic cytokines secreted by Th17 cells (IL-6, IL-17, TNF-) and anti-osteoclastogenic cytokines secreted by Th1, Th2, Tregs, and Bregs, respectively, (IFN-γ, IL-4, and IL-10) are the major players in bone remodeling under estrogen-deficient inflammatory conditions (15,19). Thus, we next analyzed the levels of these cytokines in the sera of (H) Bar graphs representing the percentage of CD4 + Rorγt + Th17 in PP. ANOVA was used to analyse the results, and then unpaired Student t-tests were used to compare the indicated groups. Values are expressed as mean ± SEM, and equivalent outcomes were observed in two independent experiments. For the indicated groups, p 0.05 (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001) was used as the cutoff for statistical significance.

CQ Skews the Cytokine Balance under Estrogen-Deficient Conditions
Osteoclastogenic cytokines secreted by Th17 cells (IL-6, IL-17, TNF-α) and antiosteoclastogenic cytokines secreted by Th1, Th2, Tregs, and Bregs, respectively, (IFN-γ, IL-4, and IL-10) are the major players in bone remodeling under estrogen-deficient inflammatory conditions (15,19). Thus, we next analyzed the levels of these cytokines in the sera of mice in all the groups. When compared to the Ovx mice group, we discovered that administering CQ significantly decreased the levels of osteoclastogenic cytokines like IL-6, IL-17, and TNFα along with significantly (p < 0.05) increasing the levels of anti-osteoclastogenic cytokines like IL-4, IL-10, and IFN-γ. (Figure 10). Taken together our data establishes that Cissus quadrangularis administration skews the cytokine balance even under estrogen-deficient conditions and thus enhances bone health.
Cells 2023, 12, x FOR PEER REVIEW 21 of 26 mice in all the groups. When compared to the Ovx mice group, we discovered that administering CQ significantly decreased the levels of osteoclastogenic cytokines like IL-6, IL-17, and TNF-α along with significantly (p < 0.05) increasing the levels of anti-osteoclastogenic cytokines like IL-4, IL-10, and IFN-γ. (Figure 10). Taken together our data establishes that Cissus quadrangularis administration skews the cytokine balance even under estrogen-deficient conditions and thus enhances bone health. Figure 10. CQ skews cytokine balance in Ovx mice : Serum samples from all the three groups viz., Sham, Ovx, and Ovx + CQ were analyzed for levels of pro and anti-inflammatory cytokines by ELISA. ANOVA was used to analyse the results, and then unpaired Student t-tests were used to compare the indicated groups. Values are expressed as mean ± SEM, and equivalent outcomes were observed in two independent experiments. For the indicated groups, p 0.05 (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001) was used as the cutoff for statistical significance.

Discussion
Over the past few years, plant-based formulations are gaining much importance with evolving pieces of evidence indicating the therapeutic role of phytoconstituents in maintaining bone-related diseases [23,38], especially via regulating the host osteoimmune system [39]. In the present investigation, we sought to elucidate the osteoprotective effect of CQ on bone health under both ex vivo and in vivo conditions. We discovered that CQ treatment significantly reduced the differentiation and functional activity of osteoclasts. Figure 10. CQ skews cytokine balance in Ovx mice: Serum samples from all the three groups viz., Sham, Ovx, and Ovx + CQ were analyzed for levels of pro and anti-inflammatory cytokines by ELISA. ANOVA was used to analyse the results, and then unpaired Student t-tests were used to compare the indicated groups. Values are expressed as mean ± SEM, and equivalent outcomes were observed in two independent experiments. For the indicated groups, p 0.05 (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001) was used as the cutoff for statistical significance.

Discussion
Over the past few years, plant-based formulations are gaining much importance with evolving pieces of evidence indicating the therapeutic role of phytoconstituents in maintaining bone-related diseases [23,38], especially via regulating the host osteoimmune system [39]. In the present investigation, we sought to elucidate the osteoprotective effect of CQ on bone health under both ex vivo and in vivo conditions. We discovered that CQ treatment significantly reduced the differentiation and functional activity of osteoclasts. Moving forward, we next investigated the osteoprotective potential of CQ under in vivo conditions. We discovered that oral treatment of CQ significantly improves bone mass by enhancing the microarchitecture of the LV-5, femoral, and tibial bones' trabecular and cortical sections (SEM and µCT data). These data are in line with the findings by Banu et al. and Ramachandran et al. which demonstrated the osteoprotective potential of CQ in the cancellous and cortical regions of femoral and proximal tibial bones in mice and Wistar rats (Banu 2012; Ramachandran 2021). In addition, a study highlighted the potential of CQ in decreasing both pain and swelling along with accelerating the healing of fractured jaws (Hemal 2015). Moreover, our histology data clearly revealed that CQ administration has no cytotoxic effect on different organs of the body, and also no change in body weight was observed after CQ administration ( Figures S2A,B and S3).
BMD is a well-established determining factor for assessing future incidences of fragility fractures in osteoporosis [40,41]. Various earlier investigations with small animal models have highlighted the bone-healing and osteoprotective properties of CQ [42,43]. The current study's findings provide additional evidence that CQ treatment enhances BMDs in both trabecular and cortical regions of the LV-5, femoral, and tibial bones. Importantly, to date no study has ever explored and correlated the osteoprotective and immunoporotic mechanisms of CQ in bone health. Therefore, the present study for the first time delineated CQ's capacity to improve bone health via modulating the host osteoimmune system.
Our group along with others has already established the pioneering role of Tregs-Th17 cells under osteoporotic conditions. Recently, we also discovered the role of Bregs in osteoporosis which further modulates the pivotal balance of Tregs and Th17 cells. These studies thus highlight and establish the strategic role of "Breg-Treg-Th17" cell axis in the therapeutic management of osteoporosis. In support of this, a recent study from our group has already reported that probiotic Bifidobacterium longum ameliorates bone loss by modulating the "Breg-Treg-Th17" axis [22]. Moving ahead, we thus explored the potential of CQ in modulating the immunoporotic "Breg-Treg-Th17" cell axis in enhancing bone health. Our flow cytometry findings demonstrated that oral treatment of CQ improve bone health even under oestrogen deficient conditions by significantly increasing antiosteoclastogenic Bregs and Tregs along with reducing the proportion of osteoclastogenic Th17 cells. It is well known that the cytokines produced by immune cells, including the anti-osteoclastogenic cytokines IFN-γ, IL-4, and IL-10 and the osteoclastogenic cytokines TNF-α, IL-6, and IL-17, influence bone remodeling [15,19]. Importantly, the immunoporotic potential of CQ was further confirmed by serum cytokine analysis, which revealed that oral administration of CQ significantly decreased the osteoclastogenic cytokines viz. IL-6, IL-17 (Th17 cells), and TNF-α, and increased the anti-osteoclastogenic cytokine, i.e., IL-10 (Bregs and Tregs), IL-4 (Th2), and IFN-γ (Th1). Collectively, the present study for the first time robustly establishes the osteo-protective and bone healing properties of CQ via modulating the "Immunoporotic" cells even under estrogen deficient inflammatory bone-loss conditions in preclinical mice models of osteoporosis ( Figure 11). Figure 11. Results summary: In PMO conditions, oral administration of CQ improves bone health by reducing the percentage of Th17 cells along with simultaneously increasing the percentages of Th1, Th2, Tregs, and Bregs immune cells in various lymphoid organs of the Ovx mice. The image was produced using Servier Medical Art, a resource made available by Servier and distributed under a Creative Commons Attribution 3.0 Unported license (https://smart.servier.com).

Conclusions
The current study clearly establishes the osteoprotective role of CQ in osteoporosis via therapeutically targeting and modulating the crucial osteoprotective "Breg-Treg-Th17" cell axis. Thus, the results of our study pave the way for future clinical employability of CQ in the management and treatment of a variety of inflammatory bone loss conditions including osteoporosis. However, more studies are still warranted to further validate

Conclusions
The current study clearly establishes the osteoprotective role of CQ in osteoporosis via therapeutically targeting and modulating the crucial osteoprotective "Breg-Treg-Th17" cell axis. Thus, the results of our study pave the way for future clinical employability of CQ in the management and treatment of a variety of inflammatory bone loss conditions including osteoporosis. However, more studies are still warranted to further validate the future perspective of CQ administration as an anabolic drug in the prevention and management of osteoporosis.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells12020216/s1, Figure S1: Cell cytotoxicity assay; Figure S2: Histological features of bone specimens; Figure S3: Histopathological analysis of kidney and lung tissues; Figure S4: Gating strategy followed for flow data analysis.  Informed Consent Statement: All applicable institutional and/or national guidelines for the care and use of animals and human samples were followed.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.