Anti-inflammatory Effects of the Triterpenoid Saponin-Enriched Fraction from She Ethnomedicine Clematis florida in a Phenol Gel-Induced Cervicitis Rat Model

doi:10.12300/j.issn.1674-5817.2025.145

Abstract

Abstract:

Objective To investigate the anti-inflammatory effects and mechanisms of the triterpenoid saponin-enriched fraction Fr.B4 from She medicine Clematis florida in a rat model of phenol-induced cervicitis. 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). After successful modeling, the C. florida treatment group received a daily oral gavage of 290 mg/kg of Fr.B4, dissolved in physiological saline, for 2 w, while the control and model groups were given an equal volume of physiological saline. ELISA was used to measure the levels of TNF-α, IL-1β, and IL-6 in rat serum and cervical tissue. Hematoxylin and eosin (HE) staining was performed to observe the pathological changes in cervical tissue. Non-targeted metabolomics 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 In the model group, rats 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<0.01). Pathological results showed thickening of the squamous epithelium, infiltration of inflammatory cells, and edema. After C. florida treatment, the cervical inflammation symptoms in rats were significantly alleviated, with a notable reduction in the levels of pro-inflammatory cytokines in serum and cervical tissue (P<0.05 or P<0.01). Cervical tissue edema decreased, inflammatory cell infiltration was reduced, and the squamous epithelium thickening was improved. Metabolomics analysis revealed 346 and 564 differential metabolites in the feces and urine of the model and control groups, respectively. After treatment with Fr.B4, 236 and 451 metabolites in the feces and urine were significantly restored, involving pathways including cAMP, AMPK, and glutathione metabolism. Anti-inflammatory metabolites like boswellic acid and oleanolic acid were increased. Gut microbiota analysis showed dysbiosis in the model group, with an imbalance in the Firmicutes/Bacteroidetes ratio. The C. florida treatment group could remodel the intestinal microbiota. The α-diversity of microbiota was similar to the control group. Specific bacterial taxa were found to correlate significantly with metabolites. Conclusion The triterpenoid saponin-enriched fraction Fr.B4 from She Medicine C. florida exerts anti-cervicitis effects by reducing pro-inflammatory cytokine levels and modulating the gut microbiota balance, and influencing metabolic pathways.

Key words: Clematis florida, Cervicitis, Gut Microbiota, Ethnomedicine, Rat

CLC Number:

R-332

ZHANG Haitao,WEI Yalan,FAN Xiaojing,et al. Anti-inflammatory Effects of the Triterpenoid Saponin-Enriched Fraction from She Ethnomedicine Clematis florida in a Phenol Gel-Induced Cervicitis Rat Model[J]. Laboratory Animal and Comparative Medicine. DOI: 10.12300/j.issn.1674-5817.2025.145.

Figures/Tables 5

Figure 1 Pathological changes in rat cervical tissues after phenol gel modeling and C. florida treatment
Note： A： Experimental timeline： After one day of phenol gel treatment， modeling rats were divided into two groups. The model group and the C. florida treatment group were respectively administered with physiological saline and Fr. B4 for 15 d； B： Cervical physiology in each group of rats； C： HE staining of cervical tissue sections and representative microscopy images （×200， scale bar = 500 μm）. Magnified images （×400， scale bar = 200 μm）.

Figure 2 Expression of inflammation-related proteins in rat serum and cervical tissue after C. florida treatment
Note： A-C and D-F respectively show the serum and cervical tissue levels of IL-1β， IL-6， and in the rats of each group detected by ELISA assay. N=10， *P<0.05， **P<0.01， ***P<0.001， ****P<0.0001.

Figure 3 Comparative analysis of feces metabolites in rats after C. florida treatment for cervicitis
Note： A：PCA of feces metabolites from each group. C （Control）， M （Model）， T （Treatment）. B：Volcano plot of differential metabolites comparing the model vs control group and the C. florida treatment vs model group. C： KEGG enrichment analysis of representative metabolites comparing the model vs control group and the C. florida treatment vs model group. D：Analysis of representative differential metabolites comparing the model vs control group and the C. florida treatment vs model group. **P<0.01，***P<0.001.

Figure 4 Comparative analysis of urinary metabolites in rats after C. florida treatment for cervicitis
Note： A：PCA of urinary metabolites from each group. C （Control）， M （Model）， T （Treatment）. B： Volcano plot of differential metabolites comparing the model vs control group and the C. florida treatment vs model group. C： KEGG enrichment analysis of representative metabolites comparing the model vs control group and the C. florida treatment vs model group. D： Analysis of representative differential metabolites comparing the model vs control group and the C. florida treatment vs model group. **P<0.01，***P<0.001.

Figure 5 Effects of C. florida on the gut microbiota of rats with cervicitis
Note： A：PCoA （Principal coordinates analysis） based on 16S rDNA from rats in the control， cervicitis model， and C. florida treatment group， using a non-metric multidimensional scaling method （left： based on weighted Unifrac distance； right： based on unweighted Unifrac distance）. B： UPGMA clustering tree analysis based on weighted UniFrac distance. C （Control）， M （Model）， T （Treatment）. C-H： α-diversity analysis using the chaos， simpson， shannon， pielou， dominance， and observed species indices， with statistical comparison performed using the Kruskal-Wallis test. I： Co-expression correlation analysis between the fecal metabolome and gut microbiota 16S rDNA groups based on Spearman’s correlation algorithm. J： Co-expression correlation analysis between the urinary metabolome and gut microbiota 16S rDNA groups based on Spearman’s correlation algorithm. *P<0.05，**P<0.01.

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