Contribution of Attenuation of TNF-α and NF-κB in the Anti-Epileptic, Anti-Apoptotic and Neuroprotective Potential of Rosa webbiana Fruit and Its Chitosan Encapsulation

Abstract

Rosa webbiana L. (Rosaceae) is one of the least reported and most understudied members of this family. It is native to the Himalayan regions of Pakistan and Nepal. The anti-convulsant effect of n-hexane extract of fruit of Rosa webbiana was investigated in a pentylenetetrazole (PTZ)-induced animal model of epilepsy. Male Sprague-Dawley rats were divided into six groups (n = 7) including control, PTZ (40 mg/kg), diazepam (4 mg/kg) and n-hexane extract (at 50, 150 and 300 mg/kg). Convulsive behavior was observed and resultant seizures were scored, animals sacrificed and their brains preserved. Chitosan nanoparticles were prepared using the ionic gelation method and characterized by UV-analysis, zeta potential and Fourier transform infrared spectroscopy (FTIR). The effects of all the treatments on the expression of phosphorylated cytokine tumor necrosis factor α (p-TNF-α) and phosphorylated transcription factor nuclear factor kappa B (p-NF-κB) expression in the cortex and hippocampus of the brains of treated rats were studied through enzyme linked immunosorbent assay (ELISA) and morphological differences and surviving neuronal number were recorded through hematoxylene and eosin (H&E) staining. Significant changes in seizures score and survival rate of rats were observed. Downregulation of neuro-inflammation, p-TNF-α and p-NF-κB was evident. Gas Chromatography-Mass Spectrometry (GC-MS) analysis of this fraction showed multiple constituents of interest, including esters, alkanes and amines.

Keywords: Rosaceae; anticonvulsant; n-hexane extract.

Figure 1

(a) Survival rate of animals (n = 5) in each group treated with PTZ, ethyl acetate, methanol and n-hexane extracts of fruit of Rosa webbiana. No animal could survive in case of ethyl acetate and PTZ, while two animals survived in methanol extract and four in n-hexane extract and diazepam. (b) The seizure score observed at 50 mg/kg, 150 mg/kg and 300 mg/kg of n-hexane extract. The data were expressed as mean ± SEM. Symbol * indicates a significant difference from the PTZ group at p < 0.05, while *** indicates significant difference at p < 0.001; symbol ^### shows a significant difference from saline at p < 0.001. (c) The cell survival fraction in MCF-7 cells (n = 3) in response to the n-hexane extract of fruit of Rosa webbiana (RW).

Figure 2

(a) DPPH scavenging by n-hexane extract of Rosa webbiana at different concentrations (b) IC₅₀ value for ascorbic acid and Rosa webbiana. No significant difference from control (ascorbic acid) could be observed.

Figure 3

(a) Chitosan-TPP NPs. (b) Chitosan-P.E. NPs.

Figure 4

Percentage of the age encapsulation efficiencies calculated at different wavelength.

Figure 5

(a) FTIR spectra of n-hexane extract of Rosa webbiana. (b) GC-MS chromatogram of the n-hexane extract of fruit of Rosa webbiana.

Figure 6

(a) Zeta potential of cs-TPP NPs. (b) Zeta potential of n-hexane cs-NPs.

Figure 7

Illustrates (a) percent survival rate of all treated groups; symbols ** and *** show the significant difference from PTZ group at p < 0.01 and p < 0.001; symbol ^### shows significant difference from NPs control at p < 0.00. (b) Seizure score of all treated groups; symbols *, ** and ^### indicate significant differences at p < 0.05, p < 0.01 (from PTZ) and p < 0.001 (from normal saline), respectively.

Figure 8

(a) Illustrates the effect of Rosa webbiana on the downregulated expression of p-NF-κB in the cortex and hippocampus of all treated rats; symbol ### symbolizes the significant difference from saline at p < 0.001, while symbols ** and *** indicate significant difference from PTZ at p < 0.01 and p < 0.001, respectively. (b) Effect of Rosa webbiana on TNF-α expression in the cortex and hippocampus of all treated rats; symbols. ^### symbolizes the significant difference from saline at p < 0.001, while symbols ** and *** indicate a significant difference from PTZ at p < 0.01 and p < 0.001.

Figure 9

Slides illustrate morphological differences in the cortex and hippocampus of the brain of treated groups of animals using hematoxylin and eosin (H&E) staining (a). The effect on the number of neurons is evident in graphs; (b,c)Symbols ### indicate significant difference from saline at p < 0.001 while symbols *, ** and *** shows significant difference from PTZ (disease) group at p < 0.05, 0.01 and 0.001 respectively.

Figure 10

Schematic description of the contribution of oxidative stress, neuro-inflammation and blood–brain barrier destruction in epileptogenesis and the protective mechanisms of Rosa webbiana, including antioxidant mechanisms, neuroprotection through reduced cytokine release (TNF-α) and downstream NF-kB inhibition and improved drug delivery.

Figure 11

(a) Rosa webbiana plant. (b) Fruit.