Therapeutic Effects Of A Cistanche-Based Herbal Formula On Primary Dysmenorrhea Via Modulation Of The COX-2/NF-κB Pathway
Jul 31, 2025
2. Methods
2.1. Oxytocin-Induced Mouse Writhing Test
Forty-eight female ICR mice were randomly divided into six groups (n = 8): control, model, ibuprofen (85.00 mg/kg), and low-, medium-, and high-dose Cistanche-based herbal formula groups (7.14, 14.28, and 28.57 mL/kg). The dosing was calculated based on the clinical human dose (100 mL/70 kg/day), converted to animal equivalents using body surface area scaling, with the herbal treatment at 5, 10, and 20 times the clinical dose.
All animals received daily oral gavage for 10 consecutive days. One hour after the final dose, oxytocin (2 U/mouse) was injected intraperitoneally to induce writhing behaviors. The latency to the first writhing response and the number of writhes within 20 minutes were recorded to assess analgesic efficacy.
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2.2. Primary Dysmenorrhea (PD) Rat Model
2.2.1. Grouping and Dosing
Forty-eight female SD rats were similarly randomized into six groups (n = 8): control, model, ibuprofen (51.00 mg/kg), and low-, medium-, and high-dose Cistanche-based formula groups (4.28, 8.57, and 17.10 mL/kg), corresponding to 0.5, 1, and 2 times the clinically equivalent dose.
2.2.2. Model Establishment
A rat model of PD was induced using subcutaneous injections of estradiol benzoate (0.5 mg on days 1 and 10; 0.2 mg from days 2 to 9) to sensitize the uterus, followed by oxytocin (2 U/rat, intramuscular) on day 11 to trigger uterine hypercontraction. On day 2, all treatment groups began oral dosing. Uterine contractility and its variation rate were measured via a tension transducer connected to a physiological signal acquisition system.
Formulas:
Uterine Contractility = Amplitude × Frequency
Variation Rate (%) = (Post-model − Pre-model) / Pre-model × 100%
2.2.3. ELISA Assays
After contractility measurements, blood was collected from the abdominal aorta, and uterine tissues were harvested. ELISA kits were used to quantify:
PGF2α, PGE2, β-EP in serum
TXB2, 6-keto-PGF1α in uterine homogenates
2.2.4. Western Blot for COX-2/NF-κB Pathway
Protein levels of COX-2, p-IKKβ, IKKβ, p-IκBα, IκBα, p-p65, p65, and β-actin were measured in uterine tissues using SDS-PAGE and Western blotting, followed by grayscale analysis via Quantity One software.
2.2.5. NO and iNOS Activity
Uterine NO levels were determined using the Griess reaction (540 nm absorbance), and iNOS enzyme activity was measured via colorimetric assay (530 nm).
2.3. Statistical Analysis
Data were expressed as mean ± SD. ANOVA or Kruskal-Wallis tests were used depending on data distribution. A p value < 0.05 was considered statistically significant.
3. Results
3.1. Effect on Oxytocin-Induced Writhing
The Cistanche-based formula significantly reduced the number of writhing episodes and prolonged writhing latency, especially at higher doses (p < 0.01), comparable to ibuprofen, indicating potent analgesic activity.
3.2. Inhibition of Uterine Hypercontractility
The model group showed significantly heightened uterine activity and variation rate (p < 0.01). Treatment with ibuprofen and all doses of the Cistanche formula significantly reduced these parameters (p < 0.01), confirming its antispasmodic effect.
3.3. Regulation of Prostaglandin Levels
The model group showed elevated PGF2α/PGE2 ratio (p < 0.01), which was significantly reduced in all Cistanche-treated groups (p < 0.01).
3.4. Reduction of TXB2 / 6-keto-PGF1α
TXB2/6-keto-PGF1α was significantly upregulated in the model group (p < 0.01), while the Cistanche-based formula lowered these levels dose-dependently (p < 0.01).
3.5. Suppression of NO and iNOS
NO and iNOS levels were significantly elevated in the model group (p < 0.05, p < 0.01, respectively). Treatment with Cistanche, particularly at medium and high doses, significantly suppressed these inflammatory mediators.
3.6. Upregulation of β-Endorphin (β-EP)
Serum β-EP, an endogenous analgesic peptide, was markedly reduced in the model group (p < 0.01). Treatment with Cistanche and ibuprofen restored β-EP levels (p < 0.05).
3.7. Modulation of COX-2/NF-κB Signaling
The model group exhibited increased expression of COX-2, p-IKKβ/IKKβ, p-IκBα/IκBα, and p-p65/p65, with reduced IκBα (p < 0.01). All Cistanche treatment groups significantly reversed these changes, suggesting inhibition of the COX-2/NF-κB inflammatory pathway.
4. Discussion
This study demonstrates that a Cistanche-centered herbal formula effectively alleviates primary dysmenorrhea through:
Pain relief (via increased β-EP)
Suppression of uterine hypercontractility
Inhibition of prostaglandin and thromboxane pathways
Downregulation of NO/iNOS
Blockade of the COX-2/NF-κB signaling axis
Mechanistically, Cistanche and its co-formulated botanicals are known for their anti-inflammatory and antispasmodic properties:
Cistanche deserticola contains echinacoside and acteoside, which exhibit neuroprotective and anti-inflammatory effects via NF-κB suppression.
Rosa rugosa (rose) contributes geraniol, which reduces oxidative stress and NO production.
Pueraria lobata (kudzu root) provides puerarin, shown to improve microcirculation and inhibit NF-κB in ischemic models.
Angelica dahurica (white angelica) includes imperatorin, which inhibits p65 phosphorylation, attenuating NF-κB activation.
These synergistic actions contribute to the downregulation of PGF2α, TXB2, NO, and iNOS, while upregulating β-EP, restoring balance in uterine inflammatory responses.
Conclusion
The Cistanche-based formula holds significant promise as a botanical therapeutic candidate for primary dysmenorrhea, with clear evidence of:
Anti-inflammatory and anti-spasmodic activity
COX-2/NF-κB pathway modulation
Endogenous pain regulation via β-endorphin
This study provides preclinical evidence supporting its development into a safe, natural, and effective herbal remedy for menstrual pain, offering an alternative to NSAIDs with potentially fewer side effects.
