Ethanol-precipitated Saccharides: Essential for Danggui Sini decoction against oxaliplatin-induced neurotoxicity

In traditional Chinese medicine (TCM), oxaliplatin-induced peripheral neuropathy (OIPN) is understood as a manifestation of "blood impediment" and "flaccidity" syndromes caused by blood deficiency and cold stagnation in the meridians. Danggui Sini Decoction (a well-known warming formula composed of seven herbs: Danggui, Guizhi, Baishao, Xixin, Gancao, Tongcao, and Dazao) is specifically indicated for these patterns. It works by nourishing blood and warming the meridians, and has been used clinically to alleviate OIPN. However, modern processing methods such as ethanol precipitation may remove insoluble components (e.g., polysaccharides), raising concerns regarding potential efficacy loss.

To investigate the impact of removing the ethanol-precipitated fraction on the neuroprotective effects and mechanisms of Danggui Sini Decoction.

The Danggui Sini Decoction extract (DSD) was fractionated via ethanol precipitation into precipitate (PAP) and supernatant (SAP), followed by compositional characterization. OIPN was induced in mice, which were then treated with DSD, PAP, or SAP. Neurotoxicity was assessed by behavioral tests, histopathology (H&E and Nissl staining) of the sciatic nerve and dorsal root ganglia (DRG), and serum levels of NT/NGF (ELISA). The gut barrier (H&E; IHC for ZO-1/occludin), abundance of Prevotellaceae_UCG-001 (qPCR), short-chain fatty acids (SCFA) levels (LC-MS), and glutathione (GSH) cycle metabolites (ELISA, LC-MS) were analyzed respectively. Cdk5/p35 pathway was evaluated both in vitro (ND7/23 cells) and in vivo by qRT-PCR and IHC.

PAP contained 90.1 % saccharides, including polysaccharides (85.5-1494.7 kDa) and a low-molecular-weight saccharide (237.0 Da). All treatments ameliorated OIPN: DSD > SAP > PAP in pain relief and nerve protection; DSD > PAP > SAP in NGF/NT restoration. All improved colonic structure (DSD > PAP > SAP) and Prevotellaceae_UCG-001 (PAP > DSD > SAP). DSD has the most ability to increase butyrate, acetate, and propionate, followed by PAP, and the worst is SAP. In addition, SAP has the most significant effect on reversing GSH depletion. In vitro, sodium butyrate (NaB), DSD, SAP, and PAP downregulated the expression of calmodulin, Camk2a, Cdk 5, and Cdk5r1. In vivo, all reduced DRG CaMKII, Cdk 5, and p35, though PAP and SAP were less than DSD.

PAP, the ethanol-precipitated saccharide fraction, is essential for DSD in the treatment of OIPN. PAP particularly regulated gut microbiota (e.g., Prevotellaceae_UCG-001) and enhanced SCFAs production (especially butyrate), indirectly suppressing Cdk5/p35 pathway via the potential GSH cycle; SAP primarily prevented GSH depletion, an effect that may be associated with direct modulation of the GSH cycle. PAP and SAP synergistically contribute to DSD on the treatment of OIPN through complementary pathways.