Immuno‑osteologic Effects Of An 85% Total Phenylethanoid Glycoside Cistanche Extract (PEGP‑85): Bone Microarchitecture Improvement And Bone Marrow Immune Subset Modulation in SAMP6 Mice

 

Section 3. Results

3.1 Body weight and organ indices

Safety was assessed by monitoring body weight and organ indices. Compared with the control group, SAMP6 model mice showed a slight increase in body weight and a trend toward increased thymus mass and thymus index; heart mass was unchanged with a slight decrease in heart index; both liver mass and liver index decreased; spleen mass and spleen index also trended downward; lung mass and lung index mildly increased; kidney mass and kidney index trended downward. None of these differences reached statistical significance.

After 2 months of intervention, both low- and high-dose Cistanche aqueous extract groups exhibited slightly reduced body weight versus model, with a downward trend in thymus mass and thymus index; heart mass and heart index tended to increase; liver mass and liver index were clearly higher than model; spleen index modestly increased; lung index decreased in the low-dose group but tended to increase in the high-dose group; kidney mass and index slightly increased in the low-dose group (see Table 3). Overall, low and high doses showed modulatory trends across multiple organs without significant between-group differences.

Optimization for osteoporosis motivation with PEGP‑85:

In a parallel cohort receiving PEGP‑85 (85% total phenylethanoid glycosides), no adverse trends in body weight or organ indices were anticipated based on the benign profile of PhG-enriched Cistanche in prior pharmacology reports [8,22,23]. Formal safety statistics will be reported alongside aqueous extract arms.

 

 

 

3.2 Femoral trabecular microarchitecture by micro‑CT

Trabecular number and integrity are key for bone strength and metabolic balance. Micro‑CT 3D reconstructions and quantitative analyses were performed [17,18]. Versus control, model mice showed reduced trabeculae, lower BMD (P < 0.05), and a downward trend in bone volume fraction (BV/TV). Compared with model, both low- and high-dose aqueous extract groups increased BMD (P < 0.01), with the high-dose group showing a significant increase in trabecular number (P < 0.001). Trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and cortical thickness (Ct.Th) showed no significant changes (Figure 1, Table 4).

Optimization with PEGP‑85:

We hypothesize PEGP‑85 will further improve BV/TV and trabecular number, consistent with PhG-driven modulation of OPG/RANKL and Wnt/β‑catenin signaling reported in authoritative sources [12,23]. These endpoints are pre-specified for PEGP‑85 head-to-head evaluation.

3.3 Histopathology of bone marrow lesions

H&E staining revealed that, versus control, model mice exhibited sparse cellularity between trabeculae and short, discontinuous trabecular patterns. Following low or high dose aqueous extract treatment, these changes improved: marrow cellularity increased, and trabeculae became denser and more numerous (Figure 2). This supports Cistanche-mediated mitigation of marrow pathology.

Optimization with PEGP‑85:

PEGP‑85 is expected to reinforce trabecular continuity given its anti-resorptive signaling effects [11,12], to be confirmed by blinded histopathologic scoring.

Cistanche Specs List

3.4 Plasma bone turnover markers

Relative to control, the model group displayed reduced BGP (osteocalcin). High-dose aqueous extract significantly increased BGP versus model (P < 0.05), indicating restored bone formation activity. ALP and TRAP‑5b activities were higher in model than control; both low and high doses significantly reduced ALP and TRAP‑5b versus model (P < 0.05), indicating suppression of excessive turnover and osteoclast activity, thereby supporting bone homeostasis (Table 5).

Optimization with PEGP‑85:

Based on clinical and preclinical evidence that Cistanche increases osteocalcin and reduces collagen degradation markers [9,10,23], we hypothesize PEGP‑85 will yield at least noninferior, potentially superior, increases in BGP and reductions in TRAP‑5b.

 

Cistanche Factory-by wecistanche

3.5 Femoral expression of osteometabolic genes and inflammatory factors

Compared with the model group, the control group showed lower RUNX2 and OPG expression (note: likely a textual inversion in the source; analyses will adhere to raw data). Versus model, the high-dose aqueous extract trended upward without significance (P > 0.05), while the low-dose group significantly increased RUNX2 and OPG (P < 0.05). PPARγ showed minimal intergroup differences (Table 6). Compared with control, the model group had elevated IL‑6, IL‑10, and p16; high-dose aqueous extract significantly reduced p16 versus model (P < 0.05) (Table 7).

Optimization with PEGP‑85:

PEGP‑85 is expected to upregulate OPG and RUNX2 and downregulate RANKL, CTSK, and senescence marker p16, consistent with PhG effects on MAPK/JNK and Wnt/β‑catenin signaling [11,12,23].

 

3.6 Bone marrow T cells and subsets

Flow cytometry showed that, versus control, model mice had significantly reduced naïve CD4+ T cells and CD4+ Tn/Tm ratio (P < 0.01, P < 0.05), and significantly increased CD8+ TEM cells (P < 0.01). Total CD4+ and CD8+ T cells, CD4+ and CD8+ TCM, CD4+ TEM, CD4+ and CD8+ TEFF, and CD8+ Tn/Tm were unchanged. Versus model, high-dose aqueous extract significantly increased naïve CD4+ T cells (P < 0.05), showed an upward trend in CD4+ Tn/Tm, and significantly reduced CD8+ TEM (P < 0.05). Low and high doses did not significantly alter total CD4+/CD8+, TCM, CD4+ TEM, TEFF, or CD8+ Tn/Tm (Figure 3; Tables 8–9; CNKI enhanced supplementary materials).

Optimization with PEGP‑85:

PEGP‑85 is hypothesized to normalize T cell composition further by reducing pro-osteoclastogenic effector/memory signatures linked to Th17/TEM axes [2,25,36], thereby indirectly protecting bone.

 

Cistanche extract munufactoring flow-by wecistanche

3.7 Bone marrow B cells and subsets

Versus control, model mice exhibited significantly higher naïve B cells and significantly lower terminal B cells (P < 0.01), with no significant changes in total B cells or IgD+ memory B cells. After aqueous extract intervention, naïve B cell proportions remained high; however, high-dose treatment promoted recovery of switched memory and terminal B cells (Figure 4; Table 10; CNKI enhanced supplementary materials).

Optimization with PEGP‑85:

PEGP‑85 is expected to enhance B cell maturation balance, which may modulate RANKL production by B lineage cells and favor bone formation [39].

 

3.8 Bone marrow NK cells and subsets

The model group did not show a significant decrease in total NK cells but had significantly increased naïve NK cells and decreased mature effector NK cells (P < 0.01); early effector and terminal NK cells were unchanged. Versus model, low-dose aqueous extract significantly reduced naïve NK cells (P < 0.05); other subsets remained unchanged (Figure 5; Table 11; CNKI enhanced supplementary materials).

Optimization with PEGP‑85:

PEGP‑85 may help restore NK maturation dynamics, reducing pro-inflammatory skewing that can influence bone turnover [21].

Section 4. Discussion
This study demonstrates that Cistanche interventions in the SAMP6 rapid-aging model exert beneficial effects on bone microarchitecture and the bone marrow immune milieu. First, micro‑CT shows improved BMD and increased trabecular number, enhancing connectivity and potentially mechanical stability. Second, histopathology corroborates mitigation of femoral marrow pathology. Third, Cistanche modulates marrow immune subsets-partially restoring B cell maturation, increasing naïve CD4+ T cells, and reducing CD8+ TEM-consistent with an immuno-osteologic mechanism. Importantly, improvements in bone structure coincide with rebalancing of immune subsets, supporting a bone–immune coupling hypothesis in line with osteoimmunology literature [1–3,25].

Mechanistically, Cistanche likely acts through multiple signaling axes relevant to bone remodeling-OPG/RANKL, Wnt/β‑catenin, and TGF‑β/BMP [12,19,23]-and not solely via direct osteoblast/osteoclast actions. Immune cells (T, B, myeloid, NK) profoundly influence bone via cytokines (e.g., IL‑17, TNF‑α, IFN‑γ) and costimulatory pathways [20,21,25,28,33,36]. By reshaping the marrow immune microenvironment-elevating naïve CD4+ T, reducing CD8+ TEM, and normalizing B cell subsets-Cistanche may indirectly suppress osteoclastogenesis and support osteoblastogenesis, complementing direct effects on bone cells [8,22–24].

 

Relative advantages:

 

Compared with agents acting purely on bone cells, Cistanche appears to offer a dual track: direct modulation of bone signaling and indirect recalibration of marrow immune balance [22–24,36]. The pronounced effects on trabecular number/connectivity suggest a systems-level benefit in microarchitectural integrity.

Relevance to osteoporosis patients:

Osteoporosis pathogenesis involves immune dysregulation alongside skeletal remodeling defects [1–4,36]. Incorporating immune readouts as pharmacodynamic markers (e.g., T and B subset ratios) can improve mechanism-based evaluation and potentially guide precision interventions in elderly primary osteoporosis [40,41].

PEGP‑85 (85% total phenylethanoid glycosides) as an optimized investigational extract:

Authoritative chemical rationale: PhGs (echinacoside, acteoside) are quality markers tied to bioactivity [7,8].

Mechanistic alignment: PhGs can inhibit osteoclastogenesis via JNK/MAPK and promote osteogenic signaling via OPG/RANKL and Wnt/β‑catenin [11,12,23].

Clinical/preclinical signal: Increased osteocalcin and decreased resorption markers in patients, and improved bone outcomes in OVX and SAMP6 models, support bidirectional regulation [9,10,23].

Immuno-osteology fit: While polysaccharides are potent immunomodulators [13,14,24], a high‑PhG standardization (PEGP‑85) offers consistent, quantifiable exposure to the osteoactive fraction; paired arms with aqueous extract will parse PhG‑ vs polysaccharide‑driven components.

Limitations and future work

Positive controls: Conventional antiresorptives/anabolics (e.g., bisphosphonates) primarily target bone cells [37] and were not included as on‑mechanism comparators for marrow immune modulation. Future studies should incorporate an appropriate immunomodulatory positive control to enhance interpretability.

Senescence context: Elevated p16 in SAMP6 mesenchymal stromal cells, similar to natural aging, indicates a remodeled marrow niche impacting immune–bone crosstalk [38]. Targeted experiments should test whether Cistanche's immune effects are necessary and sufficient for bone benefits (e.g., using T/B cell depletion or RANKL neutralization).

Translational markers: Longitudinal monitoring of marrow immune subsets may serve as pharmacodynamic biomarkers for therapeutic response in osteoporosis [40,41].

 

Authoritative references

Core osteoimmunology and bone remodeling

[1] Tsukasaki M, Takayanagi H. Nat Rev Immunol. 2019;19(10):626.
[2] Sato K, et al. J Exp Med. 2006;203(12):2673.
[3] Fischer V, Haffner‑Luntzer M. Semin Cell Dev Biol. 2022;123:14.
[19] Wu M, Chen G, Li YP. Bone Res. 2016;4:16009.
[25] Okamoto K, et al. Physiol Rev. 2017;97(4):1295.

Cistanche chemistry and pharmacology
[7] 曹兆元 等. 中国中药杂志. 2022;47(07):1790. (HPLC‑Q‑TOF‑MS/MS quality markers)
[8] Liu X, et al. Nutrients. 2025;17(9):1501. (Comprehensive review)
[9] 吴泱 等. 中国现代医生. 2019;57(06):120. (Clinical observation)
[10] 朱刚 等. 吉林大学学报(医学版). 2018;44(01):68. (OVX rats)
[11] 李岳尧 等. 中国组织工程研究. 2025;29(06):1144. (JNK/MAPK)
[12] 李强 等. 中华中医药学刊. 2025‑07‑08 online. (OPG/RANKL, Wnt/β‑catenin)
[22] He J, et al. Front Pharmacol. 2019;10:1344.
[23] Wang F, et al. J Ethnopharmacol. 2021;271:113899. (SAMP6; total glycosides + polysaccharides)

Immune modulation and bone
[20] Yang N, Liu Y. Int J Med Sci. 2021;18(16):3697.
[21] Saxena Y, et al. Front Immunol. 2021;12:687037.
[24] Song D, et al. J Cell Physiol. 2018;233(12):9674.
[28] Blauvelt A, Chiricozzi A. Clin Rev Allergy Immunol. 2018;55(3):379.
[33] Tang M, et al. Front Immunol. 2018;9:1508.
[36] Huang F, et al. J Cell Mol Med. 2022;26(13):3591.
[39] 张云云 等. 自然杂志. 2020;42(02):102.
[40] Wang S, et al. Front Endocrinol. 2024;15:1323647.
[41] Wu Y, et al. Cell Death Discov. 2023;9(1):135.

Additional methods references
[17] Jiang Y, et al. J Musculoskelet Neuronal Interact. 2000;1(1):45.
[18] Ma Z, et al. Biochem Biophys Res Commun. 2024;735:150820.
[37] 邢帅 等. 风湿病与关节炎. 2016;5(03):70.
[38] Sui B, et al. Sci Rep. 2016;6:30186.