Anti-Inflammatory Effects of the Triterpenoid Saponin-Enriched Fraction from She Medicine Clematis florida and Exploration of Its Mechanisms in a Rat Model of Cervicitis

doi:10.12300/j.issn.1674-5817.2025.145

Abstract

Abstract:

Objective To investigate the in vivo anti-inflammatory effects of the triterpenoid saponin-enriched fraction Fr.B4 from the She medicine Clematis florida (C. florida) in a rat model of phenol-induced cervicitis and to preliminarily explore its potential mechanisms of action. Methods Thirty 6–8-week-old female SPF-grade SD rats were randomly assigned to three groups: control, cervicitis model, and C. florida treatment group, with 10 rats in each group. The model and C. florida treatment groups were induced with cervicitis by vaginal injection of 25% phenol gel (once every 2 d, for a total of three injections), and the vulva and cervix were examined to assess the effectiveness of model establishment. After successful modeling, the C. florida treatment group was administered Fr.B4 at 290 mg/kg daily by gavage (dissolved in normal saline), whereas the model group received an equal volume of normal saline; both treatments continued for 2 weeks. The control group received no modeling procedure and was administered normal saline for 2 weeks. After the last intervention, fresh feces and urine samples were collected from all rats. The rats were euthanized after anesthesia with 40 mg/kg Zoletil 50, and the serum and cervical tissue samples were harvested. ELISA was used to measure the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in rat serum and cervical tissue. Hematoxylin and eosin (HE) staining was performed to observe the pathological changes in cervical tissue. Liquid chromatography-mass spectrometry was employed to analyze differential metabolites in feces and urine, followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and core metabolite analysis. 16S rDNA sequencing was used to assess the gut microbiota structure and diversity in each group. Statistical analysis was conducted using GraphPad Prism 9.0. Results Compared with the control group, rats in the model group exhibited cervical redness, swelling, and erosion, with significant increases in TNF-α, IL-1β, and IL-6 levels in both serum and cervical tissue (P<0.05 or P<0.01). Pathological examination of cervical tissue showed thickening of the squamous epithelium, infiltration of inflammatory cells, and edema. Compared with the model group, rats with C. florida treatment showed marked alleviation of cervicitis symptoms, with significantly reduced levels of pro-inflammatory cytokines in serum and cervical tissue (P<0.05 or P<0.01). Cervical tissue edema was alleviated, inflammatory cells were reduced, and squamous epithelium thickening was ameliorated. Metabolomics analysis revealed 346 and 564 differential metabolites in the feces and urine between the model and control groups, respectively. After treatment with Fr.B4, 236 and 451 metabolites in the feces and urine showed significant shifts toward control levels, involving pathways including cyclic adenosine monophosphate (cAMP) signaling, AMP-activated protein kinase (AMPK) signaling, and glutathione metabolism. Anti-inflammatory metabolites like boswellic acid and oleanolic acid were increased (P<0.01 or P<0.001). Gut microbiota analysis showed dysbiosis in the model group, with an imbalance in the Firmicutes/Bacteroidetes ratio. The Fr.B4 treatment remodeled the gut microbiota. The α-diversity of microbiota shifted toward that of the control group. Specific bacterial genera were found to correlate significantly with metabolites (P<0.05 or P<0.01). Conclusion The triterpenoid saponin-enriched fraction Fr.B4 from She medicine C. florida may exert anti-cervicitis effects by reducing pro-inflammatory cytokine levels, modulating the gut microbiota balance, and remodeling metabolism.

Key words: Clematis florida, Cervicitis, Gut microbiota, Metabolomics, SD rats

CLC Number:

R-332
R285.5

ZHANG Haitao,WEI Yalan,FAN Xiaojing,et al. Anti-Inflammatory Effects of the Triterpenoid Saponin-Enriched Fraction from She Medicine Clematis florida and Exploration of Its Mechanisms in a Rat Model of Cervicitis[J]. Laboratory Animal and Comparative Medicine, 2026, 46(5): 616-628. DOI: 10.12300/j.issn.1674-5817.2025.145.

Figures/Tables 5

Figure 1 Pathological changes in rat cervical tissues after induction of cervicitis with phenol gel and C. florida treatment

Note： A， Experimental timeline （one day after three times of phenol gel treatment， rats with successful model induction were divided into the model group and the C. florida treatment group and were administered normal saline and Fr.B4 from C. florida， respectively， by gavage for 2 consecutive weeks）； B， Vulvar phenotype of rats in each group； C， Representative light microscopy images of HE-stained cervical tissue sections （above， scale bar=500 μm） and magnified images of cervical tissue （below， scale bar=200 μm）. Arrows indicate inflammatory cell infiltration； stars indicate cellular vacuolar degeneration.

Figure 2 Expression of inflammation-related proteins in rat serum and cervical tissue detected by ELISA after treatment with the triterpenoid saponin-enriched fraction Fr.B4 from C. florida

Note： A-B， The levels of IL-1β （interleukin-1β）， IL-6 （interleukin-6）， and TNF-α （tumor necrosis factor-α） in serum and cervical tissue of rats in each group， respectively. The control group received no phenol gel treatment and was intragastrically administered normal saline for 2 weeks. The model group was intragastrically administered normal saline for 2 weeks after phenol gel treatment. The treatment group was intragastrically administered the triterpenoid saponin-enriched fraction Fr.B4 from C. florida for 2 weeks after phenol gel treatment. In each group， n=10. ^ns P>0.05， ^* P<0.05， ^** P<0.01， ^*** P<0.001， ^**** P<0.000 1.

Figure 3 Comparative analysis of fecal metabolites in rats with cervicitis after treatment with the triterpenoid saponin-enriched fraction Fr.B4 from C. florida

Note： A， Principal component analysis （PCA） of fecal metabolites from each group （the control group received no phenol gel treatment and was intragastrically administered normal saline for 2 weeks； the model group was intragastrically administered normal saline for 2 weeks after phenol gel treatment； the treatment group was intragastrically administered the triterpenoid saponin-enriched fraction Fr.B4 from C. florida for 2 weeks after phenol gel treatment）. B， Volcano plots of differential metabolites comparing the model vs control group （left panel） and the Fr.B4 treatment vs model group （right panel）. C and D， KEGG enrichment analysis of representative metabolites and the analysis of representative differential metabolites comparing the model vs control group （left panel） and the Fr.B4 treatment vs model group （right panel）. VIP， variable importance in projection； FC， fold change. ^** P<0.01，^*** P<0.001.

Figure 4 Comparative analysis of urinary metabolites in rats with cervicitis after treatment with the triterpenoid saponin-enriched fraction Fr.B4 from C. florida

Note： A， Principal component analysis （PCA） score plot of metabolites in each group （the control group received no phenol gel treatment and was intragastrically administered normal saline for 2 weeks； the model group was intragastrically administered normal saline for 2 weeks after phenol gel treatment； the treatment group was intragastrically administered the triterpenoid saponin-enriched fraction Fr.B4 from C. florida for 2 weeks after phenol gel treatment）. B， Volcano plots of differential metabolites： cervicitis model group vs control group （left panel）， and Fr.B4 treatment group vs cervicitis model group （right panel）. C and D， Representative KEGG enrichment analysis and the analysis of representative differential metabolites： cervicitis model group vs control group （left panel）， and Fr.B4 treatment group vs cervicitis model group （right panel）. VIP， variable importance in projection； FC， fold change. ^** P<0.01， ^*** P<0.001.

Figure 5 Effects of the triterpenoid saponin-enriched fraction Fr.B4 from C. florida treatment on the gut microbiota of rats with cervicitis

Note： A， Principal coordinates analysis （PCoA） of 16S rDNA sequencing data from rats in the control， cervicitis model， and Fr.B4 treatment groups （left， based on weighted UniFrac distance； right， based on unweighted UniFrac distance）. B， Unweighted pair-group method with arithmetic mean （UPGMA） clustering tree based on weighted UniFrac distance. C-H， α-diversity indices including Chao1， Simpson， Shannon， Pielou， dominance， and observed species. Statistical differences among groups were assessed using the Kruskal–Wallis test. I， Pearson correlation analysis between fecal metabolome and gut microbiota 16S rDNA data. J， Pearson correlation analysis between urine metabolome and gut microbiota 16S rDNA data. The control group received no phenol gel treatment and was intragastrically administered normal saline for 2 weeks. The model group was intragastrically administered normal saline for 2 weeks after phenol gel treatment. The treatment group was intragastrically administered the triterpenoid saponin-enriched fraction Fr.B4 from C. florida for 2 weeks after phenol gel treatment. ^* P<0.05， ^** P<0.01.

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