The effect of Zuogui-Jiangtang-Yishen decoction on the intestinal flora's response to L-α-phosphatidylcholine and L-tyrosine in patients with diabetic kidney disease: an in vitro study

Abstract

Background: Animal and cell studies have demonstrated that Zuogui-Jiangtang-Yishen decoction (ZGJTYS) has a favorable effect on the treatment of diabetic kidney disease (DKD). Our previous clinical research also showed that ZGJTYS prevents DKD in a manner similar to that of benazepril. Nevertheless, the interactions between ZGJTYS and the human gut microbiota require further investigation, particularly its interference in the intestinal flora response to food ingredients that may increase DKD risk, such as L-α-phosphatidylcholine and L-tyrosine.

Objective: The aim of this study was to evaluate the regulatory function of ZGJTYS on human gut microbiota and explore the effect of ZGJTYS on the intestinal flora response to L-α-phosphatidylcholine and L-tyrosine.

Methods: ZGJTYS was prescribed from the First Affiliated Hospital of Hunan University of Chinese Medicine. High-throughput sequencing of bacterial 16S RNA genes and fungal internal transcribed spacer (ITS) sequences was used for intestinal flora analysis. An in vitro gut microbiota simulation model was used to investigate the effect of ZGJTYS on the intestinal flora's response to L-α-phosphatidylcholine and L-tyrosine. Ultra-high-performance liquid chromatography and mass spectrometry were used for non-targeted metabolomics analysis.

Results: Compared to the control group, the microbial diversity of DKD was significantly reduced by ZGJTYS treatment; three bacterial genera, including Parabacterioids, were significantly higher; eight bacterial genera, including Prevotella_9, and the linoleic acid content were significantly lower. A receiver operating characteristic curve analysis using Parabacterioids and Prevotella_9 showed an area under the curve greater than 0.75, indicating good predictive performance. ZGJTYS intervention restored some of the normal bacterial genera, such as Rickettsia and Metarhizium, which were regulated by L-α-phosphatidylcholine and L-tyrosine. Furthermore, ZGJTYS effectively restored several significantly different Kyoto Encyclopedia of Genes and Genomes metabolic pathways related to immunity and disease to normal, such as efferocytosis and tryptophan metabolism.

Conclusion: ZGJTYS was found to effectively restore the microbiota that were altered by L-α-phosphatidylcholine and L-tyrosine to normal, along with their metabolites. However, the mechanism by which ZGJTYS exerts its preventive and therapeutic effects on DKD through the gut microbiota still requires further study.

Keywords: L-tyrosine; L-α-phosphatidylcholine; Zuogui-Jiangtang-Yishen decoction; diabetic kidney disease; in vitro fermentation; intestinal flora.

FIGURE 1

Analysis of the bacterial and fungal beta diversity in the original fecal samples. (A) and (B) are the bacterial beta diversity in the original fecal samples; (C) and (D) are the fungal beta diversity in the original fecal samples. CF represents the DKD group, HF represents the healthy college student volunteer control group, and YF represents the middle-aged and elderly non-metabolic disease control group.

FIGURE 2

Lefse analysis of the bacterial composition differences in the original fecal samples. (A) represents the significantly different bacterial genera between the CF and HF groups, (B) represents the significantly different bacterial genera between the CF and YF groups, and (C) represents the relative abundance analysis of bacterial genera that showed significant differences in both (A,B). The yellow marked bacterial genera indicate significant differences in both (A,B). CF represents the DKD group, HF represents the healthy college student volunteer control group, and YF represents the middle-aged and elderly non-metabolic disease control group.

FIGURE 3

ROC diagnostic curves based on differential bacterial genera. (A). ROC diagnostic curve generated using 11 differential bacterial genera. (B). ROC diagnostic curve generated using Parabacterioids and Prevotella_9.

FIGURE 4

Beta diversity analysis of the effects of ZGJTYS, L-α-phosphatidylcholine, and L-tyrosine on the compositions of the bacterial and fungal communities. (A,C,E) are the results of the bacterial beta diversity analysis, while (B,D,F) are the results of the fungal beta diversity analysis. CF represents the DKD group, and HF represents the healthy college student volunteer control.

FIGURE 5

Comparative analysis of alpha diversity alterations following regulatory interventions of L-α-phosphatidylcholine, L-tyrosine and ZGJTYS. (A−D) show the bacterial alpha diversity results. (E−H) show the fungal alpha diversity results.

FIGURE 6

Bubble plot of the enriched KEGG pathways and content distribution of linoleic acid. (A−C) are bubble plots of the enriched KEGG pathways, while (D−F) are comparison plots of the linoleic acid contents. The X-axis in the bubble plot represents the degree of the KEGG enrichment of substances, the Y-axis represents enriched KEGG pathways, the color on the right indicates the P-value (Pvalue) of the hypergeometric distribution test for the KEGG enrichment, and the circle size represents the number of enriched substances. CF represents the DKD group, and HF represents the healthy college student volunteer control group. (A) HF_VI vs. CF_VI; (B) HF_VI vs. HF_VI_Choline; (C) CF_VI vs. CF_VI_Choline.

FIGURE 7

Venn diagram analysis showing significantly enriched KEGG pathways following supplementation with L-α-phosphatidylcholine, L-tyrosine, and ZGJTYS. (A) Number of significantly enriched KEGG pathways for the comparisons HF_VI vs HF_VI_Choline and HF_VI vs HF_VI_ZGJTYS_Choline. (B) Number of significantly enriched KEGG pathways for the comparisons CF_VI vs CF_VI_Choline and CF_VI vs CF_VI_ZGJTYS_Choline. (C) Number of significantly enriched KEGG pathways for the comparisons HF_VI vs HF_VI_Tyrosine and HF_VI vs HF_VI_ZGJTYS_Tyrosine. (D) Number of significantly enriched KEGG pathways for the comparisons CF_VI vs CF_VI_Tyrosine and CF_VI vs CF_VI_ZGJTYS_Tyrosine. Restored pathways indicates pathways that lost significant enrichment after adding ZGJTYS. Not restored pathways indicates pathways that retained significant enrichment after adding ZGJTYS.