Danggui Sini decoction alleviates oxaliplatin-induced peripheral neuropathy by regulating gut microbiota and potentially relieving neuroinflammation related metabolic disorder

Abstract

Background: Danggui Sini decoction (DSD), a traditional Chinese medicine formula, has the function of nourishing blood, warming meridians, and unblocking collaterals. Our clinical and animal studies had shown that DSD can effectively protect against oxaliplatin (OXA)-induced peripheral neuropathy (OIPN), but the detailed mechanisms remain uncertain. Multiple studies have confirmed that gut microbiota plays a crucial role in the development of OIPN. In this study, the potential mechanism of protective effect of DSD against OIPN by regulating gut microbiota was investigated.

Methods: The neuroprotective effects of DSD against OIPN were examined on a rat model of OIPN by determining mechanical allodynia, biological features of dorsal root ganglia (DRG) as well as proinflammatory indicators. Gut microbiota dysbiosis was characterized using 16S rDNA gene sequencing and metabolism disorders were evaluated using untargeted and targeted metabolomics. Moreover the gut microbiota mediated mechanisms were validated by antibiotic intervention and fecal microbiota transplantation.

Results: DSD treatment significantly alleviated OIPN symptoms by relieving mechanical allodynia, preserving DRG integrity and reducing proinflammatory indicators lipopolysaccharide (LPS), IL-6 and TNF-α. Besides, DSD restored OXA induced intestinal barrier disruption, gut microbiota dysbiosis as well as systemic metabolic disorders. Correlation analysis revealed that DSD increased bacterial genera such as Faecalibaculum, Allobaculum, Dubosiella and Rhodospirillales_unclassified were closely associated with neuroinflammation related metabolites, including positively with short-chain fatty acids (SCFAs) and sphingomyelin (d18:1/16:0), and negatively with pi-methylimidazoleacetic acid, L-glutamine and homovanillic acid. Meanwhile, antibiotic intervention apparently relieved OIPN symptoms. Furthermore, fecal microbiota transplantation further confirmed the mediated effects of gut microbiota.

Conclusion: DSD alleviates OIPN by regulating gut microbiota and potentially relieving neuroinflammation related metabolic disorder.

Keywords: Danggui Sini decoction; Fecal microbiota transplantation; Gut microbiota; Metabolic disorder; Neuroinflammation; Oxaliplatin-induced peripheral neuropathy.

Fig. 1

DSD alleviated OIPN and the inflammatory response. A Protocols of drug administration and mechanical allodynia test, in the rat OIPN model. B Body weight measurements. C Measurement of withdrawal threshold for mechanical allodynia (von Frey test). D Representative images of Nissl-stained DRG sections (63 ×). Lightly stained cytoplasm and shrunken nucleoli (red arrow) were analyzed. Histograms showed nucleolar area. E ELISA-based detection of LPS, IL-6, and TNF-α in serum and DRG extracts. F Analysis of IL-6 and TNF-α mRNA levels in DRG by qPCR. *P < 0.05, **P < 0.01, vs. OXA group; n = 11 in the CON group; n = 10 in the OXA and DSDOXA groups, n = 9 in the ABXOXA group

Fig. 2

DSD protected against OXA-induced gut microbiota imbalance. A PCoA analysis of gut microbiota composition based on ASVs. B Phylum-level analysis of gut bacteria diversity among experimental groups. Histograms show Firmicutes to Bacteroidetes ratios. C Genus-level analysis of gut bacteria diversity among the experimental groups. D Heatmap of functional profiles of microbial communities in the CON, OXA, and DSDOXA groups. Gradient transition from blue to red reflects change in abundance, from low to high. *P < 0.05, **P < 0.01, vs. OXA group; n = 6 in every group

Fig. 3

DSD attenuated OXA-induced metabolic alterations. A Volcano plots showing differential metabolites under two comparison strategies. B Venn plot showing overlapping differential metabolites under two comparison strategies. C Bubble plots showing the most relevant pathways co-enriched by the differential metabolites. D Relative abundance of critical and prominent secondary metabolites associated with the most relevant metabolic pathways in the three groups, expressed as relative increase. E GC–MS analysis of SCFAs (acetic acid, butyric acid, valeric acid, and decanoic acid) in feces. F Heatmap of Spearman’s correlation between DSD-adjusted gut microbiota and regulated metabolites. *P < 0.05, **P < 0.01, vs. OXA group; n = 6 in every group

Fig. 4

DSD attenuated OXA-induced intestinal permeability. A Representative images of colon sections stained with H&E (20 ×). Epithelial cells (blue arrows) and the mucosal layer (black arrow) are indicated. B Representative IHC images showing the expression of tight junction-related proteins in colon tissues (20 ×). Histograms show IOD/ARE (Mean density). C Transmission electron microscopy (2K × , 10K ×) analysis of epithelial junction complex and bridge and mitochondria in colon tissue. *P < 0.05, **P < 0.01, vs. OXA group; n = 3 in every group

Fig. 5

The protective effects of DSD in OIPN are transferred by FMT. A FMT protocol. B Body weight measurements. C Measurement of withdrawal threshold for mechanical allodynia (von Frey test). D Representative images of Nissl-stained DRG sections (63 ×). Lightly stained cytoplasm and shrunken nucleoli (red arrow) were analyzed. Histograms show nucleolar area. E ELISA-based detection of LPS, IL-6, and TNF-α in serum and DRG extracts. F Alpha-diversity of gut microbiota composition. G PCoA analysis of gut microbiota composition. H Genus-level analysis of bacterial composition under two comparison strategies. I Representative IHC images of colon sections showing the expression of tight junction-related proteins (20 ×). Histograms show IOD/ARE (mean density). *P < 0.05, **P < 0.01, vs. OXA_OXA group; n = 6 in OXA_OXA group, n = 7 in DSD_OXA and DSDOXA_OXA groups

Fig. 6

Schematic illustration of the possible mechanisms that DSD-induced changes in gut microbiota ameliorate OIPN symptoms. DSD treatment beneficially regulated OXA-induced gut dysbiosis by enriching beneficial bacteria such as Faecalibaculum, Allobaculum, Dubosiella, and Rhodospirillales_unclassified. This lead to reduced intestinal permeability, decreased LPS leakage, increased SCFA levels, decreased neurotransmitters and neurotoxins, increased neuroprotective agents, alleviating DRG inflammation and hyperalgesia