Oral andrographolide loaded lipid nanocarriers alleviate stress behaviors and hippocampal damage in TNF alpha induced neuroinflammatory mice

Abstract

This study aimed to improve the delivery efficacy of andrographolide (Andro) by encapsulating it in nanostructured lipid carriers (NLCs) and to evaluate its effectiveness in reducing systemic inflammation. These AndroNLCs exhibited homogeneity with a particle size of 131.40 ± 1.30 nm and approximately 89% encapsulation efficiency. AndroNLCs potentially enhanced oral efficacy by improving gastrointestinal stability, with reduced toxicity and inflammation in SH-SY5Y neuroblastoma cells. Inflammation was induced in sexually active C57BL/6 male mice with five intraperitoneal doses of 63 µg/kg TNF-alpha every three days. This was accompanied by daily oral administration of 10 mg/kg AndroNLCs, venlafaxine, or 1 mg/kg dexamethasone for 14 days. Mice with TNF-alpha-induced inflammation showed sickness signs and abnormal behaviors, assessed via physical changes, anxiety and depression tests (i.e., open field, elevated-T maze, tail suspension, and forced swimming), and biochemical assays. These changes included weight loss and compensatory responses to inflammation, as indicated by increased immune- and stress-modulated organ weights, elevated serum corticosterone levels, altered liver function markers, and higher levels of hippocampal IL-6 and TNF-alpha. Furthermore, histological analysis showed pyknotic cells, reduced layer thickness, and decreased hippocampal cell survival. Conversely, AndroNLCs significantly improved stress- and inflammation-related markers, alleviated behavioral abnormalities, reduced liver toxicity, and restored hippocampal morphology, showing effects greater than Andro alone and comparable to traditional treatments. These findings suggest that AndroNLCs have therapeutic effects on neuroinflammation but may risk contributing to mood disorders.

Keywords: Andrographolide; Inflammation; Nanostructured lipid carrier; Stress; TNF-alpha.

Fig. 1

Physicochemical characterization of andrographolide-loaded nanostructured lipid carriers (AndroNLCs). (A) Transmission electron micrograph of AndroNLCs, (B) the stability of Blank and AndroNLCs during 3 months of storage in distilled water at 25 °C, particle size and polydispersity index (PDI), (C) the zeta potential, and (D) the stability of AndroNLCs during 4 h of treatment in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) at 37 °C. PdI was measured in distilled water.

Fig. 2

Safety and anti-inflammatory profiles in an in vitro study of Blank, natural Andrographolide (Andro), and Andrographolide-loaded nanostructured lipid carriers (AndroNLCs). (A) The percentages of cell viability in the cytotoxicity test, (B) changes in pro-inflammatory levels of IL-6, and (C) TNF-alpha in SH-SY5Y neuronal cultures exposed to lipopolysaccharides (LPS). The data are presented as mean ± SEM (n = 3 per group). *P < 0.05, ***P < 0.001 versus control, ^†††P < 0.001 versus vehicle.

Fig. 3

Physical and biochemical profiles of body and organ weights, serum and hippocampal biomarkers in male mice with TNF-alpha-induced inflammation. (A) Experimental design in the animal study, (B) starting body weight, (C) final body weight, (D) percentage of daily weight gain change, (E) relative adrenal gland weight, (F) spleen weight, (G) thymus weight, (H) serum corticosterone, (I) alanine transaminase (ALT) and (J) aspartate aminotransferase (AST) levels, (K) hippocampal interleukin (IL)-6, (L) tumor necrosis factor (TNF)-alpha and (M) malondialdehyde (MDA) levels. The data are presented as mean ± SEM (n = 5–9 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001 versus control, ^†P < 0.05,^††P < 0.01, ^†††P < 0.001 versus vehicle, and ^#P < 0.05, ^##P < 0.01,^###P < 0.001 versus Andro. Andro, Andrographolide; AndroNLCs, Andrographolide-loaded nanostructured lipid carriers; DEXA, dexamethasone; VLX, venlafaxine.

Fig. 4

Behavioral profiles of locomotor activity and anxiety- and depression-like behaviors in male mice with TNF-alpha-induced inflammation using the open-field test (OFT), elevated T-maze (ETM), tail suspension test (TST), and forced swimming test (FST). (A) Time spent in the inner zone, (B) total number of line crosses, (C) number of rearing activity in the open arena, (D) time spent in inhibitory avoidance trials at baseline, (E) trail 1, (F) trail 2, (G) time spent in the one-way escape latency test of the ETM, (H) immobility time in the TST and (I) in the FST. The data are presented as mean ± SEM (n = 9 mice per group). *P < 0.05, ***P < 0.001 versus control, ^†P < 0.05, ^††P < 0.01, ^†††P < 0.001 versus vehicle and ^#P < 0.05 versus Andro. Andro, Andrographolide; AndroNLCs, Andrographolide-loaded nanostructured lipid carriers; DEXA, dexamethasone; VLX, venlafaxine.

Fig. 5

Histological profiles of H&E-stained hippocampal subregion sections, including cornu ammonis (CA) areas CA1, CA2, CA3, CA4, and the dentate gyrus (DG) in male mice with TNF-alpha-induced inflammation. Control mice (1 A–1 F); Vehicle-treated inflamed mice (2 A–2 F); Andro-treated inflamed mice (3 A–3 F); AndroNLCs-treated inflamed mice (4 A–3 F); DEXA-treated inflamed mice (5 A–5 F); VLX-treated inflamed mice (6 A–6 F). Red arrows indicate normal neuron cells, and black arrows indicate pyknotic neuronal cells. Scale bars represent 50 μm (magnification, 40×). Andro, Andrographolide; AndroNLCs, Andrographolide-loaded nanostructured lipid carriers; DEXA, dexamethasone; PCL, pyramidal cell layer; GCL, granule cell layer; VLX, venlafaxine.

Fig. 6

The summary of the study shows the composition, which includes andrographolide (Andro), sesame oil, and glycerol monostearate (GMS). This representation demonstrates the effectiveness of Andrographolide-loaded nanostructured lipid carriers (AndroNLCs), showing low cytotoxicity and significant anti-inflammatory effects in lipopolysaccharide (LPS)-pretreated SH-SY5Y neuroblastoma cell cultures, with reduced levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha). Additionally, AndroNLCs effectively alleviated anxiety, depression, and compensatory responses to TNF-alpha challenge in male C57BL/6 mice. This was demonstrated by decreased targeted organ weights, reduced corticosterone, lower hippocampal IL-6, TNF-alpha, oxidative stress levels, and improved hippocampal morphology and pyknosis with increased cell survival, suggesting potential for mitigating neuroinflammation-induced behavioral abnormalities.