Antinociceptive activity of soy isoflavone genistein in murine models of diabetes- and nerve injury-induced painful neuropathy : involved mechanisms

Neuropathic pain occurs secondarily to CNS injury or, more commonly, in association with injury to the PNS. These injuries can be caused by tumours compressing peripheral nerves, toxins used in chemotherapy, metabolic disease (diabetes), viral disease (Herpes Zoster), severe ischemic insults, trauma and disc herniations that stretch, compress or inflame a nerve root but, despite more than 40 years of research, there are still non-effective treatments. The typical pharmacological treatments for neuropathic pain of any origin are anti-depressant, anti-convulsant or topical agents; also opiates are often ineffective. There is therefore a need for new drugs that act not only on central and peripheral neuronal component, but also on immune cells. There is great interest in soy isoflavones as alternatives to endogenous estrogens not only in hormonal pathologies, but also in inflammatory, neurodegenerative diseases and pain, so we examined the therapeutic effect of genistein, the major isoflavone in soy, against painful hypersensitivity in two models of murine peripheral neuropathy: a mononeuropathy, induced by the chronic constriction injury (CCI) of sciatic nerve, and a diabetic polyneuropathy, induced by a injection of a pancreatic -cell citotoxin, streptozotocin (STZ). Genistein, the major natural phytoestrogen isoflavone in soybean, has a weak estrogenic effect and a well-known non-specific tyrosine kinase inhibitory activity at pharmacological doses. It has 7-30 times less binding affinity for estrogen receptor (ER) α than ERβ, and is therefore devoid of unwanted severe ERα agonist side effects, such as cancer promotion. It also has effects that are independent of its estrogenic activity, including protein tyrosine kinase inhibition or down-regulation, immune system modulation and anti-oxidant activity. Finally, over the last few years, public and scientific interest in phytoestrogens has increased because of their proposed neuroprotective effects against neurodegenerative diseases, neuronal damage, cerebral stroke and ischemia. The peripheral mononeuropathy, which is based on the unilateral loose ligation of sciatic nerve, simulates the human clinical condition of chronic nerve compression, such as the one that occurs in nerve entrapment neuropathy or spinal roots irritation by a lumbar disk erniation and was induced by right sciatic nerve CCI according to Bennett and Xie; diabetes was induced by a single injection of STZ (120 mg/kg, i.p.) and both models were studied in C57BL/6J adult male mice. Neuropathies are frequently associated with allodynia (pain to innocuous stimuli) and hyperalgesia (a heightened pain response generated by a painful stimulus); we evaluated changes in paw withdrawal thresholds with Von Frey filament and Plantar test. A treatment regimen with greater clinical applicability would involve compounds that are efficacious at reversing neuropathic pain symptoms once they are established, so three days after CCI and 14 days after STZ injection, genistein was injected subcutaneously (1-30 mg/kg) daily for 11 days and over three weeks, respectively. The isoflavone repeated administration reversed in time and dose-relared fashion thermal hyperalgesia and mechanical allodynia in nerve injured mice and mechanical allodynia in diabetic mice. Surely classical estrogen receptors (ER), particularly ERβ, are involved in phytoestrogen antinociceptive activity in CCI model, in fact a specific ERβ antagonist prevented both its anti-allodynic and anti-hyperalgesic action, whereas a specific ERα antagonist was ineffective and a non-selective ER antagonist only reversed the anti-allodynic effect. It is worth bearing in mind that genistein binds ER with higher affinity for the ERβ, that are particulary present in neurons, microglia, astrocytes, Schwann cells and immune cells, but less expressed than ER in estrogen-dependent tissues. In pathological nociceptive disorders, particularly in the induction of painful peripheral neuropathy, reactive radical species are generated in excess and consequently create oxidative stress in tissues; oxidative stress is the result of an imbalance between the reactive oxygen and nitrogen species and antioxidant compounds. Antioxidant effects are implicated in the antinociceptive genistein activity, since the efficacious doses on painful hypersensitivity, simoultaneusly reversed the increase in the ROS and malondialdeyde tissue levels and increased or restored the activity of antioxidant enzymes and the ratio between reduced and oxidized glutathione content. Neuropathic pain is partially mediated by neuroinflammatory mechanisms and it also modulates local neurogenic inflammation. In fact, the release of inflammatory mediators from immune and glial cells in either peripheral and CNS may have an important role in the development and the manteinance of the neuropathic pain physiopathological processes. Genistein efficacy on pain sintomatology was due also to its neuroimmunomodulatory and anti-inflammatory properties, since in peripheral and central nervous system steps involved in pain development and trasmission (the sciatic nerve proximal to the trifurcation, the L4, L5 and L6 dorsal root ganglia, the lumbar dorsal spinal cord, at the same level, and the thalamus) the phytoestrogen reduced nuclear factor-κB, nitric oxide system and proinflammatory cytokine overactivation in both peripheral neuropathy models. In conclusion these results suggest that soy isoflavone genistein ameliorates the CCI- and diabetes-induced nociceptive hypersensitivity by its antioxidant, antiinflammatory and neuroimmunomodulatory properties and it represents a possible therapeuthic hope to treat neuropathic pain that is still now devoid of satisfactorily effective treatments.