Immuno-osteologic Rationale And Preclinical Evaluation Plan For An 85% Phenylethanoid Glycoside-enriched Cistanche Deserticola Extract (PEGP-85) in SAMP6 Mice With Osteoporosis-like Bone Loss
Dec 08, 2025
Abstract
Background: Osteoporosis is characterized by decreased bone mass and disruption of bone microarchitecture. Emerging osteoimmunology evidence shows that bone marrow immune cell subsets critically regulate the balance between osteoblast- and osteoclast-driven remodeling. Traditional Chinese Medicine (TCM) texts classify Cistanche deserticola as a superior "kidney-yang–tonifying" herb with putative bone-strengthening effects, and modern pharmacology attributes immunomodulatory, anti-inflammatory, and anti-aging activities to its polysaccharides and phenylethanoid glycosides (PhGs).
Objective: To translate TCM theory and contemporary osteoimmunology into a testable preclinical program, we propose evaluating a standardized Cistanche extract enriched to 85% total phenylethanoid glycosides (PEGP-85) on bone microstructure and marrow immune subsets in SAMP6 mice.
Methods: Using micro-CT, histology, ELISA, RT-qPCR, and multicolor flow cytometry, we will quantify PEGP-85 effects on trabecular architecture, femoral pathology, bone turnover markers (ALP, BGP/osteocalcin, TRAP-5b), osteo-metabolic and inflammatory gene expression, and marrow T/B/NK cell subsets. The study follows ethical and GLP-like procedures.
Expected significance: By integrating bone-immune readouts with classical bone endpoints, this study aims to clarify whether high-PhG Cistanche (PEGP-85) exerts dual regulation-promotion of osteogenesis and inhibition of osteoclastogenesis-potentially via OPG/RANKL and Wnt/β-catenin axes and by rebalancing marrow immune subsets, in line with osteoimmunology concepts and prior reports.
Introduction
Core pathology of osteoporosis involves both reduced bone mass and deterioration of trabecular microarchitecture. Recent advances in osteoimmunology highlight bone marrow immune cell subsets as key regulators of skeletal remodeling, influencing the coupling between osteoblasts and osteoclasts through cytokine secretion and direct cell–cell interactions [1–3]. Aberrant composition and activation states of T, B, and NK cell subsets correlate with lower bone mineral density (BMD) and compromised trabecular integrity in osteoporotic conditions [3,4]. Consequently, beyond conventional bone turnover markers, quantitative and functional profiling of marrow immune subsets should be considered as pharmacodynamic endpoints to elucidate mechanisms and evaluate efficacy of candidate therapeutics [1–4].
In TCM classics (first recorded in Shennong Bencao Jing), Cistanche deserticola is categorized as a superior-grade herb reputed to "tonify kidney-yang, enrich essence and blood, strengthen sinews and bones, and moisten the intestines." The TCM doctrine that the "kidney governs bones and engenders marrow" underpins a historical rationale for potential bone-protective effects [5,6]. Modern studies identify multiple bioactive constituents in aqueous and alcoholic extracts, prominently polysaccharides and phenylethanoid glycosides (e.g., echinacoside, acteoside/verbascoside), as well as iridoids [7,8]. Clinically, Cistanche interventions have been associated with increased osteocalcin and reduced type I collagen C-terminal telopeptide in osteoporotic patients, suggesting bidirectional modulation of bone formation and resorption [9]. In ovariectomized rat models, ethanolic extracts improved osteoporotic phenotypes [10]. Mechanistically, specific Cistanche components suppress osteoclastogenesis via JNK/MAPK pathway inhibition [11] and modulate OPG/RANKL and Wnt/β-catenin signaling to rebalance bone remodeling [12]. Immunologically, Cistanche polysaccharides activate dendritic cells and enhance humoral/cellular responses through TLR2/TLR4–MAPK–NF-κB signaling and have shown adjuvant-like properties and mitigation of cyclophosphamide-induced immunosuppression [13–16]. While most immune data derive from spleen, thymus, blood, or lymph nodes, marrow-resident subsets directly impact osteoclast–osteoblast coupling. However, whether Cistanche improves bone microstructure and modulates marrow immune subsets in the SAMP6 rapid-aging mouse model remains unclear.
To address this gap and to serve the unmet needs of osteoporosis patients, we introduce an investigational extract standardized to 85% total phenylethanoid glycosides (PEGP-85). We hypothesize PEGP-85 will enhance trabecular microarchitecture, rebalance bone turnover, and beneficially modulate marrow immune subsets toward an anti-resorptive, pro-osteogenic milieu.
85% total phenylethanoid glycosides Cistanche Extract For Bone Growth
Materials
Animals
Healthy male SAMR1 and SAMP6 mice (SPF), 5 months old, were obtained from Peking University Health Science Center [license SCXK (Jing) 2022-0009]. All procedures follow national guidelines for the care and use of laboratory animals. Animals were housed in SPF rooms at Tianjin University of Traditional Chinese Medicine (21–24 °C; 42–58% humidity; 12 h light/dark). After a 1-week acclimation, protocols were approved by the Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (TCM-LAEC2025054C1542).
Investigational product and comparators
PEGP-85: Cistanche deserticola extract standardized to 85% total phenylethanoid glycosides (e.g., echinacoside, acteoside) by HPLC-Q-TOF-MS/MS referencing quality markers in [7]; intended to maximize PhG-driven immuno-osteologic effects supported by [8,11,12].
Aqueous extract reference: Prepared as per Section 2.1 for historical comparability.
Vehicle: Physiological saline.
Cistanche benefits
Instruments
N-1210BV-WB rotary evaporator (EYELA, Japan); FD-1Plus lyophilizer (Boyikang, China); 5427R benchtop centrifuge (Eppendorf, Germany); FACSymphony A1 flow cytometer (BD, USA); Aperio CT 6 whole slide scanner (Leica, Germany); Quantum GX2 micro-CT (PerkinElmer, USA); EG1150 H embedding station, RM2235 microtome, HI1220 slide warmer, HI1210 flotation bath (Leica, Germany).
Reagents
Fixatives, solvents, and histology consumables as listed in the original protocol; TruStain FcX™ (BioLegend 156604); antibodies: Alexa Fluor 700 anti-mouse CD45 (147716), FITC CD3 (100204), PerCP/Cy5.5 CD4 (100434), APC/Fire 750 CD8a (100722), PE CD44 (103008), BV605 CD62L (104437), PE/Dazzle 594 CD19 (115554), APC CD27 (124212), BV421 IgD (405725), PE/Cy7 CD49b (103518), BV785 CD11b (101243); BV Buffer (BD 563794); NovoStart SYBR qPCR SuperMix Plus (Nearshore Bio E096); NovoScript Plus cDNA kit (Nearshore Bio E047). ELISA kits for ALP, BGP, TRAP-5b (MLbio: ml598232A, ml063317A, ml063508A).
Methods
2.1 Preparation of Cistanche aqueous extract
Cistanche decoction pieces (896 g) were soaked in 10 volumes ultrapure water for 1 h, decocted to boiling and simmered 90 min, filtered; residues decocted again in 8 volumes for 60 min; filtrates combined, passed through 200-mesh sieve, concentrated to a thick paste, vacuum-dried to yield 420 g solid (46.9% yield), stored desiccated.
2.2 Animal model, dosing, and sample collection
After acclimation, mice were randomized into four groups (n = 8/group): control (Con; SAMR1), model (Mod; SAMP6), low-dose (WECD-L), and high-dose (WECD-H). Based on a 46.9% aqueous extraction yield and a clinical crude herb dose of 9 g (human), the mouse equivalent aqueous extract doses were 548.73 mg·kg−1 (WECD-L) and 1,097.46 mg·kg−1 (WECD-H), administered by daily gavage for 2 months; controls received vehicle. Body weight was recorded weekly. On the last day, mice were anesthetized with tribromoethanol; femur and tibia were dissected. Left femurs (n = 6/group) were fixed in 10% paraformaldehyde for histology and micro-CT; right femurs were used for flow cytometry.
Note on PEGP-85 arm (optimization for osteoporosis motivation):
To test the high-PhG hypothesis grounded in [7,8,11,12], an additional cohort may receive PEGP-85 at PhG-matched doses to the aqueous extract (e.g., 200–400 mg·kg−1 PEGP-85, titrated by echinacoside/acteoside content), while maintaining identical schedules and endpoints. This arm allows head-to-head comparison of PEGP-85 versus traditional aqueous extract on bone and marrow immune outcomes.
2.3 Femoral micro-CT imaging and analysis
Fixed femurs were scanned using a Quantum GX system. After warm-up and metadata entry, specimens were positioned and scanned for 15 min under standardized parameters. Post-acquisition, images were reconstructed and analyzed for trabecular bone volume fraction (BV/TV), trabecular number (Tb.N), thickness (Tb.Th), separation (Tb.Sp), and cortical indices. Environmental conditions were kept quiet, at 15–25 °C and <80% humidity, avoiding vibration and direct sunlight.
2.4 Histopathology
Femurs were fixed in 4% paraformaldehyde for 24 h, decalcified in 10% EDTA, rinsed, dehydrated through graded ethanol, cleared in xylene, and paraffin-embedded. Transverse sections (5 μm) were prepared, deparaffinized, rehydrated, stained with hematoxylin (5–10 min) with acid alcohol differentiation and bluing, counterstained with eosin (3 min), dehydrated, cleared, and mounted with neutral resin. Digital whole-slide scans were used to assess morphology.
2.5 Plasma biochemistry
Plasma ALP, BGP (osteocalcin), and TRAP-5b were measured using commercial ELISA kits per manufacturers' instructions (MLbio).
2.6 Femoral gene expression (osteometabolic and inflammatory markers)
Femoral tissue was homogenized on ice; total RNA was extracted using chloroform/isopropanol, pelleted at 12,000 rpm, 4 °C, 15 min, washed with 75% ethanol, and dissolved. RNA quantity and integrity were assessed by spectrophotometry and agarose electrophoresis. cDNA was synthesized per kit instructions. qPCR was performed on a QuantStudio 6 Flex using SYBR mix. Cycling: 95 °C 30 s; 40 cycles of 95 °C 10 s, 60 °C 30 s. 36B4 served as internal control. Relative expression was normalized to 36B4 and expressed relative to the model group. Primers for RUNX2, OPG, and other targets were obtained from a commercial provider. Targets may include RANKL, OCN, COL1A1, CTSK, TNF-α, IL-6, consistent with osteoimmunology endpoints.
Table 1. Primer Information
| Gene Name | Primer F (5'→3') | Primer R (5'→3') |
|---|---|---|
| RUNX-2 | GCCTTCACAAACACACCAG | GGTAGTGACCTGCGGAGATT |
| OPG | CAGCTTCTTGCTTGATGGAG | AAACAGCCCCAGTGACCATTCCT |
| PPARγ | TGACGGGGTCTCGGTTTGAGG | TGGGTGGGCCAGATGGCATC |
| IL-6 | ATCCAGTTGCCTTCTTGGGACTG | TAAGCCTCCGACTTGTGAAGTGG |
| IL-10 | CAGTTATTGTCTTCCCGGCTGTA | CTATGTCGCCTTGCTTTTACTGACT |
| P16 | GCTCACATCACGGTGACCTCG | GATGTCTTGAGGTCCCGCGTCT |
| 36B4 | GCAGGTGTTCGACAACGCAG | GATGATGGAGTGTGGCCACCGGA |
2.7 Bone marrow immune subset profiling
Marrow was flushed with RPMI-1640 to prepare single-cell suspensions, filtered (100 μm), centrifuged (2,500 rpm, 4 °C, 5 min), resuspended, counted, and adjusted to (1–2) × 10^6 mL−1. Fc receptors were blocked with TruStain FcX on ice for 10 min. Cells were stained in BV buffer with the listed antibody panel on ice, protected from light, for 30 min. After washing with PBS and resuspension, samples were filtered (40 μm) prior to acquisition on a FACSymphony A1. Gating strategy: CD45+ leukocytes; T cells (CD3+ → CD4+ and CD8+; naïve/central/effector subsets via CD44/CD62L); B cells (CD19+; maturation via IgD/CD27); NK cells (CD49b+CD3−); myeloid (CD11b+). Data are reported as percentages and absolute counts where possible.
2.8 Statistics
Data are presented as mean ± SD. One-way ANOVA was used for group comparisons; LSD test for homoscedastic data, Dunnett's T3 for heteroscedasticity. P < 0.05 was considered statistically significant. Analyses were performed using GraphPad Prism 9.0.
Rationale for PEGP-85 (85% total phenylethanoid glycosides) in osteoporosis
Chemical standardization: PhGs including echinacoside and acteoside are quality markers of Cistanche and correlate with pharmacological activities [7,8]. An 85% PhG standardization provides consistent exposure and facilitates mechanistic attribution.
Osteo-immune mechanisms: PhGs and related fractions reportedly inhibit osteoclastogenesis through MAPK/JNK suppression [11] and modulate OPG/RANKL and Wnt/β-catenin pathways, favoring osteoblastogenesis and suppressing resorption [12].
Clinical signal: Reports of increased osteocalcin and decreased type I collagen degradation products in patients treated with Cistanche support dual modulation of bone turnover [9].
Immunomodulation: Cistanche polysaccharides augment dendritic cell activation and TLR2/TLR4–MAPK–NF-κB signaling [13,14], while improving indices in immunosuppression models [15]. Although PEGP-85 is PhG-focused, combined or benchmarked arms against aqueous extract can help parse PhG-driven versus polysaccharide-driven immune effects.
Table 2. Immune Cells and Antibody Markers
| Cell Type | Antibody Markers |
|---|---|
| CD4⁺ T | CD45⁺ CD3⁺ CD4⁺ CD8⁻ |
| CD8⁺ T | CD45⁺ CD3⁺ CD4⁻ CD8⁺ |
| Naïve CD4⁺ T | CD45⁺ CD3⁺ CD4⁺ CD8⁻ CD44⁻ CD62L⁺ |
| TCM CD4⁺ T | CD45⁺ CD3⁺ CD4⁺ CD8⁻ CD44⁺ CD62L⁺ |
| TEM CD4⁺ T | CD45⁺ CD3⁺ CD4⁺ CD8⁻ CD44⁺ CD62L⁻ |
| TEFF CD4⁺ T | CD45⁺ CD3⁺ CD4⁺ CD8⁻ CD44⁻ CD62L⁻ |
| Naïve CD8⁺ T | CD45⁺ CD3⁺ CD4⁻ CD8⁺ CD44⁻ CD62L⁺ |
| TCM CD8⁺ T | CD45⁺ CD3⁺ CD4⁻ CD8⁺ CD44⁺ CD62L⁺ |
| TEM CD8⁺ T | CD45⁺ CD3⁺ CD4⁻ CD8⁺ CD44⁺ CD62L⁻ |
| TEFF CD8⁺ T | CD45⁺ CD3⁺ CD4⁻ CD8⁺ CD44⁻ CD62L⁻ |
| B | CD45⁺ CD3⁻ CD19⁺ |
| Naïve B | CD45⁺ CD3⁻ CD19⁺ CD27⁻ IgD⁺ |
| IgD⁺ Memory B | CD45⁺ CD3⁻ CD19⁺ CD27⁺ IgD⁺ |
| Switched Memory B | CD45⁺ CD3⁻ CD19⁺ CD27⁺ IgD⁻ |
| Terminal B | CD45⁺ CD3⁻ CD19⁺ CD27⁻ IgD⁻ |
| NK | CD45⁺ CD3⁻ CD49b⁺ |
| Naïve NK | CD45⁺ CD3⁻ CD49b⁺ CD27⁻ CD11b⁻ |
| Early Effector NK | CD45⁺ CD3⁻ CD49b⁺ CD27⁺ CD11b⁻ |
| Mature Effector NK | CD45⁺ CD3⁻ CD49b⁺ CD27⁺ CD11b⁺ |
| Terminal NK | CD45⁺ CD3⁻ CD49b⁺ CD27⁻ CD11b⁺ |
Primary endpoints
Micro-CT: BV/TV, Tb.N, Tb.Th, Tb.Sp, cortical thickness.
Plasma: ALP, BGP (osteocalcin), TRAP-5b.
Secondary endpoints
Gene expression: OPG, RANKL, RUNX2, OCN, COL1A1, CTSK, TNF-α, IL-6.
Flow cytometry: Proportions of CD4+ and CD8+ T cells (naïve/central memory/effector via CD44/CD62L), B cell subsets (IgD/CD27 states), NK cells (CD49b+CD3−), and myeloid (CD11b+). Exploratory: ratios linked to osteoclastogenesis (e.g., Th17-related signatures per [2]) if additional markers are added.
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Hypotheses
PEGP-85 improves trabecular metrics (↑BV/TV, ↑Tb.N, ↓Tb.Sp), increases osteocalcin, and reduces TRAP-5b, indicating a shift toward formation dominance.
PEGP-85 upregulates OPG, RUNX2, and Wnt/β-catenin targets while reducing RANKL and osteoclastogenic gene CTSK.
PEGP-85 rebalances marrow immune subsets, reducing pro-osteoclastogenic T cell activity (e.g., Th17-related signals per [2]) and normalizing B cell maturation profiles.
Ethics and quality assurance
All animal work is approved (TCM-LAEC2025054C1542) and conducted under SPF conditions with standardized scanning, histology, and cytometry workflows.
Limitations and notes
Most immunomodulatory literature involves polysaccharide-rich fractions; thus, including an aqueous-extract comparator helps contextualize PEGP-85 effects.
Translational claims should await confirmatory pharmacokinetic and dose-response data for PEGP-85.
References [1–3,8] are international peer-reviewed sources (Nature Reviews Immunology, Journal of Experimental Medicine, Semin Cell Dev Biol, Nutrients), providing authoritative context.
References
[1] Tsukasaki M, Takayanagi H. Osteoimmunology: evolving concepts in bone-immune interactions in health and disease. Nat Rev Immunol. 2019;19(10):626.
[2] Sato K, Suematsu A, Okamoto K, et al. Th17 functions as an osteoclastogenic helper T cell subset that links T cell activation and bone destruction. J Exp Med. 2006;203(12):2673.
[3] Fischer V, Haffner-Luntzer M. Interaction between bone and immune cells: Implications for postmenopausal osteoporosis. Semin Cell Dev Biol. 2022;123:14.
[4] 王东, 常文举, 丁海. 骨免疫学对骨质疏松症的调控作用研究进展. 中国骨质疏松杂志. 2021;27(11):1707.
[5] 李志文, 刘元禄, 杨永菊, 等. 补肾阳中药有效成分治疗骨质疏松症作用机制研究进展. 辽宁中医药大学学报. 2023;25(10):135.
[6] 李泽辉, 徐盼瑜, 李佳珊, 等. 基于代谢组学研究肉苁蓉提取物对肾阳虚证大鼠生殖功能损伤的疗效与作用机制. 中国中药杂志. 2025;50(07):1850.
[7] 曹兆元, 刘建庭, 韩彦琪, 等. 基于 HPLC-Q-TOF-MS/MS 与网络药理学的肉苁蓉质量标志物研究. 中国中药杂志. 2022;47(07):1790.
[8] Liu X, Yang Z, Han M, et al. Bioactive Components, Pharmacological Properties, and Applications of Cistanche deserticola Y.C. Ma: A Comprehensive Review. Nutrients. 2025;17(9):1501.
[9] 吴泱, 张翔, 张鸿振, 等. 中药肉苁蓉治疗骨质疏松症及调控骨代谢组学的临床观察. 中国现代医生. 2019;57(06):120.
[10] 朱刚, 孙海斌, 徐刚. 肉苁蓉醇提物对去卵巢骨质疏松大鼠的治疗作用及其机制. 吉林大学学报(医学版). 2018;44(01):68.
[11] 李岳尧, 张民, 杨家驹. 肉苁蓉苷 A 通过 JNK/MAPK 通路抑制破骨细胞活性. 中国组织工程研究. 2025;29(06):1144.
[12] 李强, 孙毅红, 霍娇娇, 等. 肉苁蓉总苷治疗骨质疏松症的研究进展. 中华中医药学刊. 网络首发: 2025-07-08.
[13] Zhao BT, Wang SF, Tao FQ, et al. Immunomodulation of thymus by Cistanche deserticola polysaccharides analyzed via transcription-associated proteomics. Ind Crops Prod. 2025;234:18.
[14] Feng S, Yang X, Weng X, et al. Aqueous extracts from cultivated Cistanche deserticola as polysaccharide adjuvant promote immune responses via facilitating dendritic cell activation. J Ethnopharmacol. 2021;277:114256.
[15] 李进花. 肉苁蓉多糖通过调控 JAK-STAT 通路对环磷酰胺诱导的小鼠卵巢早衰的保护作用研究 [D]. 2025.
[16] Thrivikraman Nair S, Kamalasanan K, Moidu A, et al. Ethyl cellulose coated sustained release aspirin spherules for treating COVID-19: DOE led rapid optimization. Int J Biol Macromol. 2021;182:1769.
