The current study was performed to determine the therapeutic potential of 7-hydroxy-3,4-dihydrocadalin in the treatment of streptozotocin-induced painful peripheral neuropathy in rodents. In our study, dose-response curve of 7-hydroxy-3,4-dihydrocadalin acute administration presented a dose-dependent antihyperalgesic effect in the formalin test in diabetic rats. In addition, this drug showed antiallodynic activity in the streptozotocin-induced tactile allodynia model. In both tests, the antineuropathic effect was equieffective to pregabalin, a drug of first line in the treatment of neuropathic pain [40, 41]. In line with these results, chronic administration of oral 7-hydroxy-3,4-dihydrocadalin also prevented the development of mechanical hyperalgesia and allodynia in mice. In contrast with the doses used in rats, the antineuropathic activity was observed in mice with doses of 7-hydroxy-3,4-dihydrocadalin 10 times greater. Differences could be attributed to the specie used (rats versus mice) since it is well known that hepatic metabolism of mice is more accelerated than rats [42–44]. To the best of our knowledge, this is the first report about the antineuropathic activity of 7-hydroxy-3,4-dihydrocadalin. Our study agree with a previous study showing the antinociceptive effect of 7-hydroxy-3,4-dihydrocadalin in non-diabetic rats submitted to 1% formalin test and Randall-Selitto model . Taken together, data suggest that 7-hydroxy-3,4-dihydrocadalin exerts antinociceptive activity in neuropathic and inflammatory pain models. In addition, results seem to point out that 7-hydroxy-3,4-dihydrocadalin has an advantage on the rest of antineuropathic drugs used in the clinic to treat painful diabetic neuropathy as this compound not only reduces neuropathic pain intensity, but also prevents the development of this condition. In this regard, results indicate that 7-hydroxy-3,4-dihydrocadalin produced its antinociceptive effect by a glucose levels-independent mechanism as this compound was not able to modify blood glucose in an acute or chronic administration schedule.
The mechanism of the antinociceptive effect of 7-hydroxy-3,4-dihydrocadalin in neuropathic diabetic rats is unknown. However, considering that opioid, serotonergic and nitrergic systems are the most promising targets for the treatment of peripheral diabetic neuropathy or neuropathic pain by natural products [9–13], and it has been reported that 7-hydroxy-3,4-dihydrocadalin activates 5-HT1, but not opioid receptors , we decided to test if these mechanisms participated in the antineuropathic activity of 7-hydroxy-3,4-dihydrocadalin in diabetic rats. In this regard, we found that the non-selective 5-HT receptor antagonist methiothepin  was able to prevent 7-hydroxy-3,4-dihydrocadalin-induced antihyperalgesia in three-week diabetic rats. This result suggests that some of the 5-HT receptors may play a role in the antihyperalgesic effect of 7-hydroxy-3,4-dihydrocadalin in streptozotocin-induced diabetic rats. In support of this, there is evidence that activation of 5-HT1A/B/D receptors participate in the peripheral antinociceptive effect observed with 7-hydroxy-3,4-dihydrocadalin in formalin-induced inflammatory pain . The participation of 5-HT1A seems unlikely because the most of literature points out a pronociceptive role of 5-HT1A receptor in several models of neuropathic pain [46–48] including thermal hyperalgesia in streptozotocin-induced diabetic mice , notwithstanding, this has been disputed [50, 51]. On the other hand, evidence of 5-HT1B/D receptors in neuropathic pain suggests that they are linked to antinociception  and could explain the antihyperalgesic effect of 7-hydroxy-3,4-dihydrocadalin.
In the current study we also found that ODQ prevented 7-hydroxy-3,4-dihydrocadalin -induced antihyperalgesic effect in the formalin test. Since ODQ is a guanylyl cyclase inhibitor , our data suggest that antihyperalgesia observed with 7-hydroxy-3,4-dihydrocadalin in diabetic rats could be due to activation of guanylyl cyclase. Hypothesizing, this activation would lead to the synthesis of cyclic GMP, which in turn, would activate the protein kinase G and finally it could open potassium channels to hyperpolarize the nociceptive neurons, as with the natural products that activate this enzyme [12, 13].
On marked contrast, subcutaneous administration of the non-selective opioid receptor antagonist naltrexone did not prevent the antihyperalagesic effect induced by the acute administration of 7-hydroxy-3,4-dihydrocadalin. This result indicates that opioid receptors do not play a role in the antihyperalgesic effect of 7-hydroxy-3,4-dihydrocadalin in streptozotocin-induced diabetic rats of 3 weeks. The lack of effect of naltrexone is not due to the dose used in this work, since this dose has been reported to be effective in previous studies in diabetic rats with similar characteristics [30, 54]. Taken together, results suggest that 7-hydroxy-3,4-dihydrocadalin exerts its antihyperalgesic affect through activation of 5-HT1 receptors and guanylyl cyclase enzyme, but not opioid receptors.
Additionally to the described mechanisms, 7-hydroxy-3,4-dihydrocadalin decreased the malondialdehyde concentration in diabetic rats to baseline values in non-diabetic rats. Our data agree with evidence demonstrating a potent scavenging activity of 7-hydroxy-3,4-dihydrocadalin on diphenyl-p-picrylhydrazyl radical or microsomal and mitocondrial lipid peroxidation [19–21]. Furthermore, 7-hydroxy-3,4-dihydrocadalin has shown to be effective to protect red cells against oxidative haemolysis  or mitochondrial enzyme activity against oxidative stress . Antioxidant activity of 7-hydroxy-3,4-dihydrocadalin results of interest as pathogenesis of diabetic neuropathy is related with metabolic changes in several pathways that interact in a mutually facilitatory fashion to cause an imbalance in the mitochondrial redox state and an increment in the expression of pro-inflammatory genes producing neuronal hypersensitivity [3, 5]. Remarkably, 7-hydroxy-3,4-dihydrocadalin has also demonstrated anti-inflammatory properties on the croton oil-induced edema test in mouse ear  and carrageenan-induced paw edema model . Speculating, 7-hydroxy-3,4-dihydrocadalin could be useful to treat the diabetic neuropathy in a more integrative manner as it combines antineuropathic, antioxidant and possibly anti-inflammatory properties.
Although pregabalin is clinically considered as the gold standard for the treatment of painful diabetic neuropathy in human beings , its use is limited due to side effects such as sedation, dizziness and somnolence that affects motor coordination [56, 57]. For this reason, equi-effective doses of 7-hydroxy-3,4-dihydrocadalin and pregabalin were compared in the rota-rod test. Interestingly, pregabalin, but not 7-hydroxy-3,4-dihydrocadalin, affected motor coordination in diabetic rats. Our results seem to point out that notwithstanding the similar antineuropathic activity between both drugs, 7-hydroxy-3,4-dihydrocadalin might present an advantage on pregabalin in the diabetic neuropathy treatment since 7-hydroxy-3,4-dihydrocadalin did not produce motor side-effects. However, further studies are necessary to assess the safety of oral acute and chronic administration of 7-hydroxy-3,4-dihydrocadalin. In addition, a clinical study of efficacy and safety study in patients with painful diabetic neuropathy is required to evaluate the real therapeutic potential of this compound in the diabetic neuropathy.