The Sanbi Decoction alleviates intervertebral disc degeneration in rats through intestinal flora and serum metabolic homeostasis modulation

Intervertebral disc degeneration (IVDD) is an essential cause of low back pain (LBP), the incidence of which has risen in recent years and is progressively younger, but treatment options are limited, placing a serious economic burden on society. Sanbi decoction (SBD) is an important classical formula for the treatment of IVDD, which can significantly improve patients' symptoms and is a promising alternative therapy. The aim of this study is to investigate the safety and efficacy of SBD in the treatment of IVDD and to explore the underlying mechanisms by using an integrated analytical approach of microbiomics and serum metabolomics, as well as by using molecular biology. A rat IVDD puncture model was established and treated by gavage with different concentrations of SBD, and clean faeces, serum, liver, kidney, and intervertebral disc (IVD) were collected after 4 weeks. We assessed the safety by liver and kidney weighing, functional tests and tissue staining, the expression of tumor necrosis factor-alpha (TNF-ɑ), interleukin 1β (IL-1β) and interleukin 6 (IL-6) inflammatory factors in serum was detected by ELISA kits, and X-ray test, magnetic resonance imaging (MRI) examination, immunohistochemistry (IHC), western blotting (WB), hematoxylin-eosin (HE) staining and safranin O-fast green (SO/FG) staining were used to assess the efficacy. Finally, we performed 16S rRNA sequencing analysis on the faeces of different groups and untargeted metabolomics on serum and analyzed the association between them. SBD can effectively reduce the inflammatory response, regulate the metabolic balance of extracellular matrix (ECM), improve symptoms, and restore IVD function. In addition, SBD can significantly improve the diversity of intestinal flora and maintain the balance. At the phylum level, SBD greatly increased the relative abundance of Patescibacteria and Actinobacteriota and decreased the relative abundance of Bacteroidota. At the genus level, SBD significantly increased the relative abundance of Clostridia_UCG-014, Enterorhabdus, and Adlercreutzia, and decreased the relative abundance of Ruminococcaceae_UCG-005 (P<0.05). Untargeted metabolomics indicated that SBD significantly improved serum metabolites and altered serum expression of 4alpha-phorbol 12,13-didecanoate (4alphaPDD), euscaphic acid (EA), alpha-muricholic acid (α-MCA), 5-hydroxyindoleacetic acid (5-HIAA), and kynurenine (Kyn) (P<0.05), and the metabolic pathways were mainly lipid metabolism and amino acid metabolism. This study demonstrated that SBD can extensively regulate intestinal flora and serum metabolic homeostasis to reduce inflammatory response, inhibit the degradation of ECM, restore IVD height and water content to achieve apparent therapeutic effect for IVDD.