Neuroprotective properties of Withametelin and Ajugarin-I against neuropathic pain and multiple sclerosis

Abstract

In the present study, we investigated the neuroprotective properties of Withametelin (WMT) and Ajugarin-I (Aju-I) against animal models of neuropathic pain and multiple sclerosis. Natural products have been studied in the preclinical models of neuropathic pain and multiple sclerosis and represent promising candidates for drug development. Withametelin, a potential phytosterol/withanolide, obtained from Datura innoxa, has excellent neuroprotective properties. Correspondingly, Ajugarin-I isolated from Ajuga bracteosa exerts significant neuroprotective effects. Initially, the neuroprotective potential of WMT and Aju-I has been confirmed against hydrogen peroxide (H2O2)-induced PC12 cells, HT22, and BV-2 cell lines. To develop potential candidates for neuropathic pain treatment, experimental models including vincristine (VCR)-induced neuropathy, streptozotocin (STZ)-induced diabetic neuropathic pain, and partial sciatic nerve ligation (PSNL)-induced neuropathic pain models have been established. The goal of this study was to explore the anti-neuropathic pain activity and underlying mechanisms of WMT and Aju-I in experimental models of neuropathic pain. The current investigation provided convincing evidence that the WMT and Aju-I treatment exhibits anti-neuropathic pain activity against the VCR-, STZ-, and PSNL-induced pain hypersensitivity. Inflammatory cytokines, oxidative stress, and genotoxicity were significantly reduced by WMT and Aju-I. They inhibited the VCR-, STZ-, and PSNL-induced histopathological changes in the sciatic nerve and spinal cord. VCR- and STZ-induced alterations in the biochemical composition of the sciatic nerve's myelin sheath were inhibited by WMT. In the spinal cord, WMT significantly reduced the expression of TRPV1/TRPM8/P2Y nociceptors, as well as MAPK signaling (ERK/JNK/p-38). Correspondingly, Aju-I significantly suppressed oxidative stress, inflammation, and apoptosis by regulating Nrf2/NF-κB and Bcl2/Caspase expression levels in VCR-treated mice. In the STZ-induced model, WMT downregulates MAPK/NF-κB signaling in the spinal cord. In the same way, the activation of the PSNL-induced NF-κB inflammatory axis was suppressed after WMT treatment. Similarly, Aju-I upregulated the expression of Nrf2 and downregulated the expression of TRPV1/TRPM8 signaling in the spinal cord STZ-induced rats. After confirmation of anti-neuropathic pain activity, an in vivo murine model of MS such as experimental autoimmune encephalomyelitis (EAE) has been established. The behavior results indicated that EAE-induced clinical score, neuropathic pain behavior, and motor xxv impairment were considerably reduced by WMT and Aju-I post-immunization treatment. They substantially reduce EAE-induced splenomegaly, spinal deformity, and elevated circulating leucocytes. They significantly reduced the extravasation of Evans blue and FITC in the brain. They markedly restored the CNS histological changes caused by EAE. WMT considerably increased the antioxidant defense mechanism in the CNS by increasing the expression of Nrf2/HO-1 while decreasing the expression of keap-1/iNOS. WMT effectively diminished neuroinflammation in the CNS by lowering TLR4/NF-κB/AP-1 immunoreactivity. Similarly, Aju-I markedly attenuated EAE-induced inflammation in the CNS by reducing the expression levels of MAPK/NF-κB. It suppressed EAE-induced apoptosis in the CNS by regulating Bax/Bcl-2/Caspase-3 expression. Additionally, the computational analysis showed significant binding interaction of WMT and Aju-I with protein targets via hydrogen and hydrophobic bonds. Thus, based on the present findings, it is concluded that WMT and Aju-I are potential novel candidates for the treatment of neuropathic pain and multiple sclerosis. Key words: Neuropathic pain, Multiple sclerosis, EAE, Vincristine, Streptozotocin, Withametelin, Ajugarin-I