Improvement of pyridoxine-induced peripheral neuropathy by Cichorium intybus hydroalcoholic extract through GABAergic system

Abstract

Pyridoxine (vitamin B6) toxicity is a well-known model for peripheral neuropathy. GABA and glutamate are two neurotransmitters in neural pathways involved in the peripheral neuropathy. Cichorium intybus (Chicory) contains glycosides and triterpenoids, which inhibit glutamatergic transmission and enhance GABAergic transmission. The present study was aimed at studying the effect of chicory extract (CE) on the pyridoxine-induced peripheral neuropathy with a particular focus on glutamatergic and GABAergic systems. In this experimental study, a high dose of pyridoxine (800 mg/kg, i.p.) was injected for 14 days to induce neuropathy in male rats. To evaluate the behavioral symptoms, three tests including rotarod, hot plate, and foot fault were used. After the induction of neuropathy, CE (50 mg/kg i.p.) was injected intraperitoneally for 10 consecutive days. Morphologically, the sciatic nerve and the DRG neurons were evaluated in the control, neuropathy, and chicory groups by H&E staining. For evaluating the mechanism, picrotoxin (1 mg/kg) and MK-801 (0.1 mg/kg) were also individually injected 15 min before the extract administration. The concentration of TNF-α in rat sciatic nerve and DRG neurons were also measured by enzyme-linked-immunoassay (ELISA). Morphological and physiological changes occurred in the DRG and sciatic nerve following pyridoxine intoxication. The CE exerted an anti-neuropathic effect on the sciatic nerve and DRG neurons and also decreased reaction time in hot plate test (p < 0.05), increased balance time in rotarod test (p < 0.001), and improved foot fault performance (p < 0.01). Moreover, CE administration reduced TNF-α level in DRG (p < 0.001) and sciatica nerve (p < 0.001). Picrotoxin, unlike MK-801, showed a significant difference in all three behavioral tests and reduced TNF-α content in comparison with group received extraction alone (with p < 0.001 for all three tests). Our results showed beneficial effects of CE on pyridoxine-induced peripheral neuropathy. Modulating of the GABAergic system mediated by TNF-α may be involved in the anti-neurotoxic effect of CE.

Keywords: CE; DRG; GABA; Glutamate; Peripheral neuropathy; Pyridoxine; Sciatic nerve.

Fig. 1

Dorsal of the lumbar segment contains DRG neurons (a) and site of the sciatic nerve (b)

Fig. 2

GC–MS chromatogram of the methanolic extract of chicory root

Fig. 3

Images of the rats suffering from neuropathy induced by pyridoxine toxicity for 14 days. They were unable to stand or walk on all four paws

Fig. 4

Pyridoxine-induced neuropathy and investigation of the role and probable mechanism of CE in the rotarod test. a High dose of pyridoxine significantly decreased rotarod time compared to control (ctrl) group (*p < 0.001). b In the treatment group, CE increased rotarod time in comparison with saline group (NS) (*p < 0.001). MK-801 had no significant effect while picrotoxin (picro) decreased rotarod time compared to the CE group (#p < 0.001). Data are shown as mean ± SEM (N = 7 per group)

Fig. 5

Pyridoxine-induced neuropathy and investigation of the role and probable mechanism of CE in foot fault test. a Foot fault increased in the pyridoxine group compared to the control (ctrl) group (*p < 0.001). b CE had a beneficial effect to reduce fault number compared to saline (NS)-treated group (*p < 0.01). The effect of MK-801 was not significant compared to the CE-treated group while picrotoxin (picro) could increase the number of foot fault in comparison with CE-treated group significantly (#p < 0.001). Data are shown as the mean ± SEM (N = 7 per group)

Fig. 6

Pyridoxine-induced neuropathy and investigation of the role and probable mechanism of CE in hot plate test. a Reaction time to heat increased in pyridoxine group compared with the control (ctrl) group. *p < 0.001. b The rats receiving CE decreased reaction time in comparison with saline (NS) group (*p < 0.05). Picrotoxin (picro) treatment group increased the duration of response to heat but Mk-801 had no significant effect (#p < 0.001). Data are shown as the mean ± SEM (N = 7 per group)

Fig. 7

Cross sections of dorsal root ganglion neurons (DRG) from control (a), rats received a high dose of pyridoxine (b), rats treated with CE (c), and the chart of DRG cells number (d). There are two types of cells in DRG neurons: A type or B type. Type A cells have a large size and less intensely central nucleolus (arrow in a). B cells are characterized by a nucleus containing basophilic and many smaller chromatin condensations (arrow in b). The result of the cell count showed that the number of A-cells are more abundant than B-cells in the control group. However, in the neuropathic group compared to the control, the number of B-cells increased significantly (*p < 0.001). CE treatment abolished the effect of pyridoxine toxicity and led to an increase in the number of A-cells (#p < 0.001) (H&E magnification 200×, scale bar 50 µm)

Fig. 8

Cross (a–c) and longitudinal (d–f) sections of sciatic nerve from control, neuropathy, and CE-treated groups, respectively. In the control group, the number of nerve fibers in both cross and longitudinal sections is greater than the neuropathy group. In chicory-treated rats, the number of nerve fibers increased and also endoneurium areas appeared smaller than the pyridoxine-treated animals. (H&E magnification 100× (a–c), scale bar 100 µm; H&E magnification: 200× (d–f), scale bar 50 µm)

Fig. 9

Measurement of TNF-α level in the DRG (a) and sciatic nerve (b). Protein levels were determined by ELISA. Data are shown as the mean ± SEM (N = 5 per group). *p < 0.001 NP vs. ctrl (a and b); **p < 0.001 NP + CE vs. NP (a and b); #p < 0.001 (a) and p < 0.01, (b) NP + CE + picro vs. NP + CE (a and b)

Fig. 10

Diagram of chicory mechanism stimulation and inhibition