Targeting Anti-Inflammatory Pathways to Treat Diabetes-Induced Neuropathy by 6-Hydroxyflavanone

Abstract

It is evident that inflammation and metabolic syndrome instigated by diabetes mellitus can precipitate diabetes-induced neuropathy (DIN) and pain. In order to find an effective therapeutic method for diabetes-related problems, a multi-target-directed ligand model was used. 6-Hydroxyflavanone (6-HF) carrying anti-inflammatory and anti-neuropathic pain potential due to its quadruplicate mechanisms, targeting cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), and opioid and GABA-A receptors was investigated. The anti-inflammatory potential of the test drug was confirmed utilizing in silico, in vitro, and in vivo tests. A molecular simulation approach was utilized to observe the interaction of 6-HF with the inflammatory enzyme COX-2 as well as opioid and GABA-A receptors. The same was confirmed via in vitro COX-2 and 5-LOX inhibitory assays. In vivo tests were performed to analyze the thermal anti-nociception in the hot-plate analgesiometer and anti-inflammatory action in the carrageenan-induced paw edema model in rodents. The potential anti-nociceptive effect of 6-HF was evaluated in the DIN model in rats. The Naloxone and Pentylenetetrazole (PTZ) antagonists were used to confirm the underlying mechanism of 6-HF. The molecular modeling studies revealed a favorable interaction of 6-HF with the identified protein molecules. In vitro inhibitory studies revealed that 6-HF inhibited the COX-2 and 5-LOX enzymes significantly. The 6-HF at dosages of 15, 30, and 60 mg/kg substantially reduced heat nociception in a hot plate analgesiometer as well as carrageenan-induced paw edema in rodent models. The authors discovered that 6-HF had anti-nociception properties in a streptozotocin-induced diabetic neuropathy model. According to the findings of this study, 6-HF was demonstrated to diminish inflammation caused by diabetes as well as its anti-nociception effect in DIN.

Keywords: 6-hydroxyflavanone; diabetes-induced neuropathy; inflammation.

Figure 1

Chemical structure of 6-HF.

Figure 2

Experimental protocol for the evaluation of 6-HF in DINP model. STZ (50): Streptozotocin 50 mg/kg, GP: Gabapentin 75 mg/kg, 6-HF: 6-hydroxyflavanone at doses of 15, 30, and 60 mg/kg administered intraperitoneally (i.p.) n = 8.

Figure 3

In silico binding of 6-HF with the cyclooxygenase-2 (COX-2) enzyme protein, showing complete interactions with the side chains of the binding sites.

Figure 4

In silico binding of 6-HF with the (A) kappa, (B) mu, and (C) delta opioid receptors, showing complete interactions with the side chains of the binding sites.

Figure 5

In silico binding of 6-HF with the GABA-A receptors: (A) 2D structure, with green color showing the hydrogen bond formation representing the ALA residue (ALA-161) of the α-subunit seen in the 4COF and the hydroxyl group of 6-HF with the side chain of the SER-159 residue in the F-subunit chain of the GABA-A receptor. (B) 3D structure, showing interaction of 6-HF with GABA-A receptors.

Figure 6

Anti-nociceptive activity of 6-HF at 15, 30, and 60 mg/kg in the hot plate test. *** p < 0.001 (MP-5) and ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 (6-HF) compared to vehicle control animals (ANOVA followed by Dunnett’s post hoc test, n = 8).

Figure 7

Effect of (a) naloxone (1.0 mg/kg; NXL-1) and (b) pentylenetetrazole (10 mg/kg; PTZ-10) on the anti-nociceptive effect of 6-HF in the mouse hot plate test. ** p ˂ 0.01, *** p ˂ 0.001 compared to morphine (5.0 mg/kg; MP-5) or 6-HF (15, 30, and 60 mg/kg) (ANOVA followed by Tukey’s multiple comparison post hoc test, n = 8).

Figure 8

Anti-inflammatory activity of 6-HF at 15, 30, and 60 mg/kg in the carrageenan-induced paw edema test. * p < 0.05, and *** p < 0.001 compared to vehicle control animals. Two-way repeated-measure ANOVA post hoc Bonferroni analysis (n = 8).

Figure 9

Induction of diabetes in streptozotocin-induced diabetic neuropathic pain model. *** p ˂ 0.001 compared to control rats (ANOVA followed by Dunnett’s post hoc test, n = 8).

Figure 10

The percent response of female rats post-administration (0, 5,15,21, and 28 d) of streptozotocin: (a) static/dynamic allodynia and (b) static/dynamic vulvodynia.

Figure 11

Effect of treatment with 6-HF at doses of 15 mg/kg (6-HF-15), 30 mg/kg (6-HF-30), and 60 mg/kg (6-HF-60) and positive control gabapentin (GP) at a dose of 75 mg/kg (GP-75) on the expression of diabetes-induced mechanical static allodynia (decrease in paw-withdrawal threshold; PWT in g) and dynamic allodynia (decrease in paw-withdrawal latency; PWL in s) in the hind paw of rats. (a) Effect of 6-HF and GP on the expression of diabetes-induced static allodynia. (b) Effect of 6-HF and GP on the expression of diabetes-induced dynamic allodynia. ^### p < 0.001 (GP-75) and ** p < 0.01, and *** p < 0.001 compared to streptozotocin-treated control animals. Two-way repeated-measure ANOVA post hoc Bonferroni analysis (n = 8).

Figure 12

Effect of treatment with 6-HF (6-HF) at doses of 15 mg/kg (6-HF-15), 30 mg/kg (6-HF-30), and 60 mg/kg (6-HF-60) and positive control gabapentin (GP) at a dose of 75 mg/kg (GP-75) on the expression of diabetes-induced mechanical static vulvodynia (decrease in the flinching-response threshold; FRT in g) and dynamic vulvodynia (decrease in the flinching-response latency; FRL in s) in the vulva of rats. (a) Effect of 6-HF and GP on the expression of diabetes-induced static vulvodynia. (b) Effect of 6-HF and GP on the expression of diabetes-induced dynamic vulvodynia. ^### p < 0.001 (GP-75) and ** p < 0.01, and *** p < 0.001 compared to streptozotocin-treated control animals. Two-way repeated-measure ANOVA post hoc Bonferroni analysis (n = 8).