Black tea rich in polygonatum sibiricum polysaccharides inhibits osteoporosis in ovariectomized mice through immune regulation and gut microbiota suppression

Abstract

There is a growing interest in identifying dietary strategies to prevent osteoporosis. Black tea has emerged as a potential candidate due to its demonstrated effects on bone metabolism. However, the presence of caffeine in black tea may have an adverse impact on bone health. In our study, we have formulated a compound black tea beverage (CBT) with reduced caffeine content and supplemented with food additives such as Polygonatum polysaccharide. This study aims to investigate alternative mechanisms underlying the anti-osteoporotic effects of this composite black tea preparation. Mice were randomly assigned to sham (S) group, OVX group, and CBT group. After surgery, statistical differences (P < 0.05) became evident when the thermal pain threshold was reached at 5 weeks, while the grip and mechanical pain thresholds were achieved at 6 weeks, persisting until 10 weeks. Following CBT intervention, IL-17 A, TNF-α, TGF-β, β-CTX and PINP levels displayed improvements (P < 0.05). Compared to the OVX mice, CBT mice exhibited varying degrees of improvement in BV/TV, Tb.Th, Tb.N, Tb.Sp (P < 0.05) and a significant decrease in RANKL and TNF-α protein expression in the bone tissue (P < 0.001), along with a significant increase in OPG and TGF-β1 protein expression (P < 0.001). Those treated with CBT exhibited various degrees of improvement in Th17 cells, Treg cells, and the Treg/Th17 cell ratio (P < 0.05) and displayed higher expressions of FOXP3 and lower expressions of ROR-γt in the spleen tissue (P < 0.05). CBT treatment was found to decrease the relative abundance of norank_f_Maurbaculaceae and Lactobacillus, while increasing the relative abundance of Lachnospiraceae NK4A136_group and Dubosella. CBT has been shown to functional behavioral assessments and bone microstructure, thereby effectively retarding the progression of osteoporosis. This impact is postulated to stem from certain bioactive constituents within CBT that act via immune regulation and gut microbiota modulation.

Keywords: Black tea; Gut microbiota; Osteoporosis; Ovariectomized mice; Polygonatum polysaccharide; immune regulation.

Fig. 1

(A-C) Changes in terms of grip strength, thermal pain threshold and mechanical pain threshold in three groups of mice before and after 10 weeks of intervention (D-I) Levels of serum IFN-γ, IL-10, RANKL, IL-17 A, TNF-α, and TGF-β after 10 weeks of behavioural monitoring. n = 12, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2

(A-B) Changes in bone metabolism biomarkers, bone microstructure and related protein expression in three groups of mice after CBT intervention. (C-D) Representative images of ABH staining (40× scale bar: 1000 μm, 100× scale bar: 400 μm) (E) Analysis of the distal femoral epiphyseal region of mice using micro-computed tomography (CT). (F-G) Representative images of Immunohistochemistry of RANKL and OPG. (H-K) Comparison of BV/TV, Tb.Th, Tb.N, Tb.Sp. (L) Protein blot analysis of bone tissue targeting proteins associated with the RANKL/OPGpathway and inflammatory factors. (M-P) Expression of RANKL, TNF-α, OPG, and TGF-β1 proteins in bone tissues. n = 12, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3

(A) Flow cytometry quantification of cell numbers in three groups with CD25 + FOXP3+/CD4 + staining. (B) Flow cytometry quantitative analysis of cell numbers in three groups, IL-17 A+/CD4 + staining. (C-E) Expression ratios of Th17, Treg, and Treg/Th17 in the spleen. (F-H) Blotting analysis of Th17 and Treg cell-associated transcriptional proteins in splenic tissues, changes in the expression ratios of FOXP3/β-catin and ROR-γt/β-catin among the three groups. n = 12, *P < 0.05, ***P < 0.001.

Fig. 4

(A-E) Comparison of Sobs, ACE, Chao, Shannon, and Simpson index values in α-diversity analysis (F) PCoA analysis based on the Bray-Curtis distance algorithm (G) NMDS analysis (Fig. 4H-I) Partial Least Squares Discriminant Analysis (PLS-DA) at the OTU and Genus levels. n = 6, *P < 0.05.

Fig. 5

(A-C) Relative abundance analyses of bacterial communities at the phylum, family and genus levels. (D) Venn diagrams were constructed for the Sham, OVX and CBT groups (E) LEfSe identified specific bacterial phenotypic variations (LDA > 3.0) (F) LDA score plots (LDA > 3.0) (G) Intestinal flora species (LDA > 4). n = 6, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6

(A-C) Spearman’s correlation analysis of gut microbiota at phylum, family and genus level with serum factors IFN-γ, IL-10, RANKL, IL-17 A, TNF-α and, TGF-β.(D) PICRUSt2 analysis identifies the top 10 KEGG pathways.

Fig. 7

(A) Schematic diagram of the effect of CBT intervention in ovariectomized osteoporotic mice; (B) Main and possible mechanism of CBT extract of improving bone metabolism through Treg/Th17 cell balance; (C) Main and possible mechanisms of improving bone metabolism through intestinal microecological balance.