Duhuo Jisheng Mixture attenuates neuropathic pain by inhibiting S1PR1/P2Y₁R pathway after Chronic Constriction Injury in mice

Background: The pathogenesis of neuropathic pain is complex and lacks effective clinical treatment strategies. Medical plants and herbal extracts from traditional Chinese medicine with multi-target comprehensive effects have attracted great attention from scientists.

Purpose: To investigate the pharmacological active components and mechanism underlying the anti-neuralgia effect of classic analgesic formulas Duhuo Jisheng Mixture (DJM).

Study design and methods: Chronic Constriction Injury (CCI) surgery was used to assess the efficiency of DJM in treating neuropathic pain. Forty-two C57BL/6 mice were evenly divided into seven groups: sham-operated, sham treatment with DJM (20 ml/kg), CCI treatment with DJM (0, 10, 20, or 40 ml/kg) and CCI treatment with Morphine (3 mg/kg). DJM irrigation stomach was implemented from post-operative day (POD) 0 to POD 21. Paw withdrawal threshold and Thermal latency were conducted on POD 0, 1, 3, 5, 7, 14 and 21. On POD 14, eEPSP in spinal dorsal horn neurons was recorded by patch-clamp. mRNA transcription in spinal cord was also monitored to screen molecular targets and signaling pathways on POD 14. To further validate the influence of DJM on the S1PR1 signaling pathway, 1 mg/kg W146 was infused into mice intrathecally and delivered to the cerebral spinal fluid through one 30 G needle with a whole 20 μl intervertebral between the L5 and L6. Furthermore, the active ingredients of DJM were identified through LC/MS equipment and predicted through Molecular Docking.

Results: We demonstrated that DJM treatment reversed CCI-induced pain sensitivity and the excitability of neurons, decreased up-regulation of astrocyte S1PR1, inhibited astrocyte activation, and further inhibited the expression of Purinergic P2Y₁R and its downstream molecule p-JNK in mouse spinal cord, as well as the release of inflammatory factors. Interestingly, due to the regulatory role of astrocytes on neurons, the effects of DJM on astrocytes ultimately manifest in pain-effector neurons, resulting in decreased p-ERK, p-CREB, and pain-marking protein c-fos in neurons. S1PR1 Antagonist W146 possessed an equivalent analgesic effect as DJM and inhibited S1PR1 and c-fos expression. According to LC/MS analysis results, a total of 33 active ingredients were screened, of which 20 active ingredients had good binding activity with S1PR1 and were considered to be the main active ingredients for analgesia.

Conclusion: This study is the first to clarify the effect and molecular mechanism of DJM for anti-neuralgia in particular, which confirms the clinical value of DJM in relieving neuropathic pain. Furthermore, this study innovatively identifies the potential pharmacological components of DJM through LC/MS and Bioinformatics technology, which forms a framework for people's understanding of DJM treatment for neuropathic pain and provides a sufficient theoretical basis of DJM for clinical application.