Therapeutic Effects Of Cistanche On Chronic Prostatitis Via STAT3/HIF-1α/Glycolysis Axis: An Experimental Study
Jul 21, 2025
Results
3.1 In Vivo Analysis
3.1.1 Effects of Cistanche-Based Treatment on General Condition in EAP Rats
During the experiment, rats in the control group showed shiny fur, active behavior, and normal diet and activity levels. In contrast, rats in the model group exhibited dull fur and lethargy, indicating successful model establishment. After Cistanche-based treatment, the fur quality and mental state of the rats improved noticeably.
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3.1.2 Effects of Cistanche-Based Treatment on Water Intake and Urinary Output in EAP Rats
Water intake and urinary output are critical indicators of health in rats. After the final day of treatment, rats were housed in metabolic cages for 24-hour measurements.
Results:
The model group showed significantly reduced urinary output compared to the control group (P < 0.05), with marginal reductions in water intake (not statistically significant).
Medium- and high-dose Cistanche groups demonstrated a significant increase in urinary output (P < 0.01), while water intake showed improvement without statistical significance.
Table 1: Comparison of 24-Hour Urinary Output and 24-Hour Water Intake in Rats (Mean ± SD, n = 7)
| Group | Dosage (mg·kg⁻¹) | Urinary Output (mL) | Water Intake (mL) |
|---|---|---|---|
| Control | -- | 19.36 ± 6.57 | 18.75 ± 1.88 |
| Model | -- | 11.59 ± 4.66* | 15.45 ± 6.16 |
| Pusitai | 34.2 | 17.88 ± 3.48 | 14.08 ± 3.04 |
| Cistanche (Low) | 197.2 | 15.50 ± 3.95 | 10.63 ± 3.20 |
| Cistanche (Med) | 591.5 | 21.56 ± 4.02** | 15.72 ± 3.01 |
| Cistanche (High) | 1774.4 | 22.54 ± 4.83** | 16.03 ± 2.84 |
*Significant differences compared to the control group: *P < 0.05.
**Significant differences compared to the model group: **P < 0.01.
3.1.3 Effects of Cistanche-Based Treatment on Prostate Index and Spleen Index
The prostate index reflects prostate inflammation and glandular structure, while the spleen index indicates systemic immune response.
Results:
The prostate index was significantly reduced in the model group compared to the control group (P < 0.05). Treatment with Cistanche significantly improved the prostate index (P < 0.05 or P < 0.01).
The spleen index increased significantly in the model group (P < 0.01), indicating heightened immune burden. Cistanche treatment significantly reduced the spleen index (P < 0.01).
Table 2: Comparison of Prostate Index and Spleen Index in Rats (Mean ± SD, n = 7)
| Group | Dosage (mg·kg⁻¹) | Prostate Index (%) | Spleen Index (%) |
|---|---|---|---|
| Control | -- | 10.57 ± 4.98 | 18.75 ± 1.88 |
| Model | -- | 7.00 ± 1.15* | 11.56 ± 1.66** |
| Pusitai | 34.2 | 9.04 ± 1.91 | 14.36 ± 0.84 |
| Cistanche (Low) | 197.2 | 8.96 ± 1.89 | 13.91 ± 1.93 |
| Cistanche (Med) | 591.5 | 11.51 ± 2.58* | 12.63 ± 3.21** |
| Cistanche (High) | 1774.4 | 11.95 ± 1.34** | 12.53 ± 2.81** |
*Significant differences compared to the control group: *P < 0.05.
**Significant differences compared to the model group: **P < 0.01.
3.1.4 Effects of Cistanche-Based Treatment on Serum Cytokines
ELISA was used to measure IL-8, IL-1β, and IgG levels in rat serum, reflecting inflammation and immune responses.
Results:
IL-8 and IL-1β levels were significantly elevated in the model group compared to the control group (P < 0.01). Cistanche treatment significantly reduced IL-8 and IL-1β levels in the medium- and high-dose groups (P < 0.01 or P < 0.05).
IgG levels were significantly higher in the model group (P < 0.05). The high-dose Cistanche group significantly lowered IgG levels (P < 0.01), suggesting modulation of immune responses and reduced immune complex formation.
Table 3: Comparison of IL-8, IL-1β, and IgG in Rat Serum (Mean ± SD, n = 7)
| Group | Dosage (mg·kg⁻¹) | IL-8 (pg·mL⁻¹) | IL-1β (pg·mL⁻¹) | IgG (pg·mL⁻¹) |
|---|---|---|---|---|
| Control | -- | 128.54 ± 12.63 | 19.64 ± 2.46 | 8.94 ± 1.51 |
| Model | -- | 197.91 ± 20.07** | 32.57 ± 3.42** | 13.14 ± 4.46* |
| Pusitai | 34.2 | 133.55 ± 10.51** | 27.32 ± 2.91* | 9.93 ± 1.61 |
| Cistanche (Low) | 197.2 | 181.26 ± 20.67 | 30.29 ± 2.77* | 13.56 ± 1.91 |
| Cistanche (Med) | 591.5 | 152.84 ± 16.32** | 27.32 ± 2.91** | 10.33 ± 1.94 |
| Cistanche (High) | 1774.4 | 139.25 ± 14.17** | 25.59 ± 2.84** | 9.45 ± 1.67** |
*Significant differences compared to the control group: *P < 0.05.
**Significant differences compared to the model group: **P < 0.01.
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3.1.5 Histopathological Changes in Prostate Tissue
HE staining results showed the following:
Model Group: Severe lymphocytic infiltration, glandular structural damage, and glandular atrophy.
Cistanche-Treated Groups: Improved glandular structure, reduced inflammatory cell infiltration, and restored tissue integrity.
These findings confirm the anti-inflammatory and tissue-protective effects of Cistanche in chronic prostatitis.
3.1.6 Expression of MINPP1 and HK in Prostate Tissue
Immunohistochemical analysis revealed:
HK Protein: The model group showed significantly increased HK expression compared to the control group (P < 0.01). After treatment with Cistanche, HK expression was significantly reduced (P < 0.01).
MINPP1 Protein: The model group exhibited significantly reduced MINPP1 expression compared to the control group (P < 0.01). Cistanche treatment significantly increased MINPP1 expression (P < 0.01).
These findings suggest that Cistanche may inhibit glycolysis by downregulating HK and upregulating MINPP1, thereby alleviating chronic prostatitis.
3.1.7 Expression of p-STAT3 and HIF-1α in Prostate Tissue
Western blot analysis showed:
p-STAT3: Expression was significantly increased in the model group compared to the control group (P < 0.01). Cistanche treatment (low and high doses) significantly reduced p-STAT3 expression (P < 0.01).
HIF-1α: Expression was significantly elevated in the model group compared to the control group (P < 0.01). Cistanche treatment (low and high doses) significantly reduced HIF-1α expression (P < 0.01).
Table 5: Comparison of p-STAT3 and HIF-1α Protein Expression in Prostate Tissue of Rats (Mean ± SD, n = 5)
| Group | Dosage (mg·kg⁻¹) | p-STAT3 (OD) | HIF-1α (OD) |
|---|---|---|---|
| Control | -- | 0.514 ± 0.03 | 0.624 ± 0.06 |
| Model | -- | 1.094 ± 0.09** | 0.884 ± 0.13** |
| Cistanche (Low) | 197.2 | 0.814 ± 0.04** | 0.674 ± 0.08** |
| Cistanche (High) | 1774.4 | 0.514 ± 0.02** | 0.645 ± 0.05** |
**Significant differences compared to the control group: **P < 0.01.
**Significant differences compared to the model group: **P < 0.01.
3.2 Metabolomics Analysis
3.2.1 Quality Control and Metabolite Profiling
QC Samples: Quality control samples clustered closely, indicating reliable data quality (Figure 4A).
PLS-DA Analysis: Clear separation was observed between the control group and the model group, confirming successful model establishment. A distinct separation was also observed between the model group and the high-dose Cistanche group, indicating therapeutic improvement (Figure 4B).
Model Validation: The PLS-DA model showed high reliability (R²Y = 0.94) and no overfitting, confirming accurate predictions (Figure 4C).
Fig.4 PCA score plot (A), PLS-DA score plot (B), and model validation (C) of samples from each group
3.2.2 Differential Metabolite Analysis
Using VIP > 1 and P < 0.05 as criteria:
Control vs. Model: Identified 85 upregulated and 108 downregulated metabolites.
Model vs. High-Dose Cistanche: Identified 84 upregulated and 100 downregulated metabolites.
Overall, 96 significantly altered metabolites were identified across the three groups (Figure 5).
3.2.3 Pathway Enrichment Analysis
Metabolic pathway enrichment analysis revealed that Cistanche treatment primarily affected:
Terpenoid and steroid biosynthesis.
Glycolysis/gluconeogenesis.
Pyruvate metabolism.
Oxidative phosphorylation.
Arachidonic acid metabolism.
Key Metabolites: Cistanche influenced glycolytic intermediates such as α-D-glucose, glycerate-2,3P₂, glycerate-2P, and pyruvate, thereby regulating glycolytic pathways (Figure 6).
Table 5: Comparison of Protein Expression of p-STAT3 and HIF-1α in Prostate Tissue of Rats (Mean ± SD, n = 5)
| Group | Dosage (mg·kg⁻¹) | p-STAT3 (OD) | HIF-1α (OD) |
|---|---|---|---|
| Control | -- | 0.514 ± 0.03 | 0.624 ± 0.06 |
| Model | -- | 1.094 ± 0.09** | 0.884 ± 0.13** |
| Cistanche (Low) | 197.2 | 0.814 ± 0.04** | 0.674 ± 0.08** |
| Cistanche (High) | 1774.4 | 0.514 ± 0.02** | 0.645 ± 0.05** |
Notes:
Significant differences compared to the control group: P < 0.01.
Significant differences compared to the model group: P < 0.01.
Discussion
Chronic prostatitis involves complex mechanisms, with immune and metabolic dysregulation playing significant roles. This study demonstrated the therapeutic effects of Cistanche in improving chronic prostatitis through the STAT3/HIF-1α/glycolysis axis.
Key Findings
Anti-Inflammatory Effects: Cistanche significantly reduced inflammation markers (IL-8, IL-1β) and immune complex formation (IgG).
Metabolic Regulation: Cistanche suppressed glycolysis by downregulating HIF-1α and HK while upregulating MINPP1.
Histological Improvements: Reduced inflammatory infiltration and restored prostate tissue integrity.
Mechanistic Insights
STAT3/IL-17 Axis: Cistanche inhibited STAT3 phosphorylation, reducing IL-17-mediated inflammation and Th17 differentiation.
HIF-1α and Glycolysis: By suppressing HIF-1α and HK, Cistanche alleviated hypoxia and metabolic disorders, breaking the "inflammation-hypoxia-glycolysis" vicious cycle.
