Ipriflavone and Ipriflavone loaded albumin nanoparticles reverse lipopolysaccharide induced neuroinflammation in rats

Abstract

Background: Neuroinflammation causes neurodegenerative conditions like Alzheimer's disease (AD). Ipriflavone (IP), therapeutic compound to postmenopausal osteoporosis, has limited estrogenic activity and is accounted as AChE inhibitor. The developing of drug delivery systems to enable drug targeting to specific sites increases the drug therapeutic effect.

Objective: The aim of the present study was to formulate and evaluate ipriflavone loaded albumin nanoparticles (IP-Np) along with free ipriflavone against lipopolysaccharide (LPS) induced neuroinflammation in rats.

Methods: Neuroinflammation was induced by intra-peritoneal (i.p) injection of LPS (250 μg/kg rat body weight) then treatments were conducted with (1) ipriflavone at two doses 50 mg/kg and 5 mg/kg, (2) IP-Np (5 mg ipriflavone/kg) or (3) IP-Np coated with polysorbate 80 (IP-Np-T80) (5 mg ipriflavone/kg). The alteration of the inflammatory response in male adult Wistar rats' brain hippocampus was investigated by examining associated indices using biochemical and molecular analyses.

Results: A significant upsurge in inflammatory mediators and decline in antioxidant status were observed in LPS-induced rats. In one hand, ipriflavone (50 mg/kg), IP-Np and IP-Np-T80 ameliorated LPS induced brain hippocampal inflammation where they depreciated the level of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and enhanced antioxidant status. In another hand, ipriflavone at dose (5 mg/kg) didn't show the same therapeutic effect.

Conclusion: The current study provides evidence for the potential neuroprotective effect of ipriflavone (50 mg/kg) against LPS-induced neuroinflammation in rats through its anti-inflammatory and antioxidant activities. Moreover, nanoparticles significantly attenuated neuroinflammation in concentration lower than the effective therapeutic dose of free drug ten times.

Fig 1. TEM images of ipriflavone loaded nanoparticles (IP-Np) at different magnifications.

Green lines illustrate particle size (nm).

Fig 2. Cumulative release of ipriflavone from nanoparticles (IP-Np) and nanoparticles coated with polysorbate 80 (IP-Np-T80).

The drug release was investigated in the dissolution mediums of pH 2.0 (simulated gastric fluid, SGF) and pH 7.4 (simulated blood fluid, SBF and simulated intestinal fluid, SIF).

Fig 3. Alterations in oxidative stress parameters.

(A) brain and serum TBARS levels, (B) brain NO, (C) GSH levels and GST and (D) GPx and SOD activities in the different experimental groups. Data represented as mean ± SD and p value is statistically significant at (***p≤0.001, **p≤0.01, *p≤ 0.05) compared to induced group and (#p≤ 0.05) compared to mock-treated group.

Fig 4. Variations in brain amyloidogenic markers (Aβ, IDE) and advanced glycation end-products.

(A) AGEs, (B) Aβ, and (C) IDE levels in the brain of different experimental groups. Data represented as mean ± SD and p value is statistically significant at (***p≤0.001, **p≤0.01, *p≤ 0.05) compared to induced group and (#p≤ 0.05) compared to mock-treated group.

Fig 5. Alterations in brain (hippocampus) of acetylcholinesterase (AChE).

A) Activity of AChE, B) expression profile of genes by reverse transcriptase (RT-PCR) of LPS-induced and treated groups versus control groups. Data represented as mean ± SD and p value is statistically significant at (***p≤0.001, **p≤0.01, *p≤ 0.05) compared to induced group and (#p≤ 0.05) compared to mock-treated group.

Fig 6. Alterations in brain inflammatory markers.

(A) TNF-α, (B) IL-6, (C) IL-1β and (D) iNOS levels in the brain of different experimental groups. Data represented as mean ± SD and p value is statistically significant at (***p≤0.001, **p≤0.01, *p≤ 0.05) compared to induced group and (#p≤ 0.05) compared to mock-treated group.

Fig 7. Alterations in gene expression of brain APP processing genes and changes in gene expression and protein levels of inflammatory transcription factor NF-κb.

A) BACE-1 and APP, B) ADAM 10 and ADAM 17, C) NF-κB p65 and D) Representative Histogram of relative intensity of protein levels of NF-κB p65 of LPS-induced and treated groups versus control groups. Data represented as mean ± SD and p value is statistically significant at (***p≤0.001, **p≤0.01, *p≤ 0.05) compared to induced group and (#p≤ 0.05) compared to mock-treated group.

Fig 8. Alterations in brain (hippocampus) protein levels of p38-MAPK and phosphorylated p38-MAPK of LPS-induced and treated groups versus control groups.

A) Representative western blotting analysis of p38-MAPK and p-p38-MAPK proteins. B) Representative Histogram of relative intensity of p38-MAPK and p-p38-MAPK using β-actin as internal control. Data represented as mean ± SD and p value is statistically significant at (***p≤0.001, **p≤0.01, *p≤ 0.05) compared to induced group and (#p≤ 0.05) compared to mock-treated group.

Fig 9. Schematic Diagram for the possible effect of ipriflavone and ipriflavone nanoformulation on LPS brain inflammation induced rats.

Ipriflavone (50mg/kg), IP-Np and IP-Np-T80 ameliorated LPS induced brain inflammation in hippocampal region of adult male rats. Neuroprotective effect can be attributed to its anti-inflammatory activity and its ability to decrease the level of pro-inflammatory cytokines and its antioxidant activity.