Comparative effects of raw and processed cistanche glycosides on the HPT axis and gut microbiota in a rat model of kidney-yang deficiency

Abstract

Introduction: Kidney Yang Deficiency (KYD) is a metabolic disorder associated with kidney damage. Its slow progression means that causative factors and effective therapeutic agents remain unclear. Extensive evidence links KYD to gut microbiome metabolic diseases and the Hypothalamic-Pituitary-Thyroid (HPT) axis. Cistanche deserticola (CD) is a commonly used traditional Chinese medicine for treating KYD. However, the precise interactions between gut microbiota and KYD, as well as the mechanisms of raw and processed CD total glycosides (CDG) in modulating KYD, require further investigation. This study aims to evaluate the effects and mechanisms of CDG in a KYD rat model using 16S rRNA gene sequencing and fecal metabolomics.

Methods: CDG was extracted from both raw and processed CD and analyzed via HPLC. Propylthiouracil-induced KYD rats were used to assess pharmacological effects, including serum levels of T₃, T₄, TSH, TRH, FFA, LPL, and NO; organ indices of the spleen, kidney, and thymus; blood cAMP/cGMP levels; and liver levels of glycogen, SDH, Ca²⁺-ATPase, and Na⁺-K⁺-ATPase. Immunohistochemistry was also performed.

Results: Fecal non-targeted metabolomics identified 98 metabolites associated with KYD, while 16S rRNA sequencing revealed 13 key intestinal microbiotas linked to KYD. CDG therapy effectively alleviated KYD symptoms by modulating the gut microbiota, improving metabolic and microbial imbalances in KYD. RG/WG significantly improves KYD rats mainly through the relationship between the intestinal microbiota and arachidonic acid metabolism. The key bacterial genera lleibacterium and Streptococcus observed in the changes of intestinal flora and fecal metabolite content were significantly negatively correlated with phosphatidylcholine and phosphatidylethanolamine.

Discussion: This integrative approach of gut microbiome and fecal metabolomics not only provides a scientific basis for CDG's preventive effects on KYD via the HPT axis but also elucidates the potential mechanisms underlying CDG's action against KYD.

Keywords: Cistanche deserticola; HPT axis; feces metabolomics; gut microbiota; total glycosides.

FIGURE 1

RG and WG symbolic constituent chromatograms. (A) RG chromatogram. (B) WG chromatogram. (C) Chromatogram of Echinacoside. (D) Chromatogram of Cistanoside A. (E) Chromatogram of Verbascoside. (F) Chromatogram of Isoverbascoside. (G) Chromatogram of 2′-Acetylacteoside. Observation wavelength (λ) = 330 nm.

FIGURE 2

The impact that CDG has on body temperature, body weight, organ index, serum, plasma, liver homogenate related to energy metabolism index cytokine. (A) Random body weight of rats at 30 days. (B) Random body temperature of rats at 30 days. (C) Thymus indexes. (D) Spleen indexes. (E) Kidney indexes. (F) Serum T₃ levels. (G) Serum T₄ levels. (H) Serum TRH levels. (I) Serum FFA levels. (J) Serum LPL levels. (K) Serum NO levels. (L) Serum TSH levels. (M) Plasma cAMP level. (N) Plasma cGMP level. (O) Plasma cAMP/cGMP level. (P) Concentration of energy metabolism index cytokine glycogen. (Q) Concentration of SDH. (R) Concentration of the Ca²⁺-ATPase. (S) Concentration of Na⁺-K⁺-ATPase.MEAN values ± SD (n = 6) were utilized to display the data. The student’s test was employed for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.005 in comparison to the CN group. In contrast to the MD group, ^# P < 0.05, ^## P < 0.01, ^### P < 0.005.

FIGURE 3

Effect of CDG on (A) H&E staining and immunohistochemical expression of (D) TPO, (E) TG and (F) TSHR proteins in the thyroid of KYD model rats. Scale bar = 50 μm, original magnification 50×. (B) Expression of TRH mRNA in the hypothalamus (C) TSH mRNA expression in pituitary gland of rats in each group.

FIGURE 4

Analysis of fecal samples’ metabolism. (A) In the positively charged ionic type of stool samples, a typical TIC of the QC, CN, MD, WG, RG, and PS groups. (B) Plots of the five groups’ PLS-DA scores according to their fecal metabolism characteristics. (C) Plots of the CN and MD groups’ OPLS-DA scores. (D) The OPLS-DA model’s S-plot of the CN and MD group. As indicated by the red dot, VIP >1.0. (E) Fecal OPLS-DA permutation test. (F) Plot of the Feces Volcano. Variable metabolite upregulation is indicated by red. Variable metabolic downregulation is indicated by blue. Metabolites with no statistical difference are shown in gray.

FIGURE 5

Path evaluation of KYD metabolites that have been considerably altered. (A) The degree of change is indicated in upregulation and downregulation on the hierarchical grouping heat map of the fecal differing metabolites. (B) Visual examination of the concentration route of modified chemicals in feces. (C) Examination of the fecal route of common metabolites in exposure to KYD. A metabolic route is represented by each circle.

FIGURE 6

Network of potential biomarker changes based on KYD and CDG regulation. Arrows point upward or downward. WG was marked by blue, and RG by green.

FIGURE 7

The variations in each group’s gut microbes. (A) The Shannon index used to determine the diversity of bacteria. (B) The Simpson index utilized for assessing variation in bacteria. (C) Bacterial richness estimated based on the Chao 1 value (D–F) Veen diagram of ASV clustering: The value inside the ASV in this area represents the number of ASVs. CN, MD, WG, RG and PS groups were marked brown, red, blue, green, yellow, respectively. (J) PCoA analysis. (H) The phylum level mean proportions of bacterial species. (I) Phylum level characterization of the microbiota in the gut by bacterium species. (G) Genus level characterization of the intestinal microbes by bacterium species. (K) Biodiversity cladogram produced by LEfSe study. (L) Taxonomic scores from LDA (log10) (score>3), the changes of differential bacteria in each group (n = 6). (M) Ratio of Firmicutes-to-Bacteroidetes and abundance of the key differentiated gut microbiota at genus level. Compared with CN group, *P < 0.05, **P < 0.01. Compared with MD group, ^# P < 0.05, ^## P < 0.01.

FIGURE 8

The following associations were found: (A) various metabolites and HPT axis ELISA indicators (the red/blue colors showed a positive/negative correlation); (B) differential metabolites and intestinal flora; (C) possible fecal metabolites mediating the possible relationship between intestinal flora and HPT axis ELISA indicators.