ANTIOXIDANT AND ANTI-INFLAMMATORY EFFECTS OF RHAMNAZIN ON LIPOPOLYSACCHARIDE-INDUCED ACUTE LUNG INJURY AND INFLAMMATION IN RATS

Abstract

Background: Acute Lung Injury (ALI) results into severe inflammation and oxidative stress to the pulmonary tissue. Rhamnazin is a natural flavonoid and known for its antioxidant and anti-inflammatory properties.

Materials and methods: The antioxidative and anti-inflammatory properties rhamnazin were tested for protection against the acute lung injury. We investigated whether rhamnazin improves the lipopolysaccharide (LPS)-induced ALI in an animal model (rat). We also studied the probable molecular mechanism of action of rhamnazin. Rhamnazin was injected intraperitoneally (i.p.) (5, 10 and 20 mg/kg) two days before intratracheal LPS challenge (5mg/kg). The changes in lung wet-to-dry weight ratio, LDH activity, pulmonary histopathology, BALF protein concentration, MPO activity, oxidative stress, cytokine production were estimated.

Results: The results showed a significant attenuation of all the inflammatory parameters and a marked improvement in the pulmonary histopathology in the animal groups pretreated with rhamnazin. The rhamnazin pretreated group also showed activation of Nrf2 pathway and attenuation of ROS such as H₂O₂, MDA and hydroxyl ion. These results indicated that rhamnazin could attenuate the symptoms of ALI in rats due to its strong antioxidant and anti-inflammatory properties.

Conclusion: The results strongly demonstrated that rhamnazin provides protection against LPS-induced ALI. The underlying mechanisms of its anti-inflammatory action may include inhibition of Nrf2 mediated antioxidative pathway.

Keywords: BALF; acute lung injury; cytokine; flavonoid; inflammation.

Figure 1

Effect of rhamnazin pretreatment on (A) lung wet/dry ratio, (B) lung tissue LDH level and (C) protein concentration in the bronchoalveolar lavage fluid. Data are presented as the mean ± standard error (n=10). *P<0.05 compared with the LPS-induced ALI group; #P<0.05 compared with the vehicle-treated ALI group. ALI, acute lung injury; LPS, lipopolysaccharide; LDH, lactate dehydrogenase.

Figure 2

Effect of rhamnazin pretreatment on pulmonary histopathology. (A) phosphate-buffered saline-treated healthy control group, (B) LPS-induced ALI group, (C) vehicle-treated ALI group and (D) 5 mg/kg rhamnazin pretreated ALI group (E) 10 mg/kg rhamnazin pretreated ALI group, and (D) 20 mg/kg rhamnazin pretreated ALI group.

Figure 3

Effect of rhamnazin pretreatment on LPS-induced pro-inflammatory cytokine secretion in the serum of differentially treated ALI mice. Levels of (A) TNF-α, (B) IL-6, (C) MIP-2 and (D) IL-1β in the serum.

Figure 4

Effect of rhamnazin on LPS-induced pro-inflammatory cytokine secretion in the BALF of differentially treated ALI mice. Levels of (A) TNF-α, (B) IL-6, (C) MIP-2 and (D) IL-1β in the BALF.

Figure 5

Effect of rhamnazin pretreatment on MPO (myeloperoxidase) activity and neutrophil accumulation in lung tissues. (A) MPO activity in the lung tissue homogenates of differentially treated ALI mice. (B) Neutrophil count in the lung bronchoalveolar lavage fluid of differentially treated ALI mice. Data are presented as the mean ± standard error (n=10). *P<0.05 compared with the LPS-induced ALI group; #P<0.05 compared with the vehicle-treated ALI mice.

Figure 6

Effect of rhamnazin pretreatment on oxidative stress. The levels of (A) H₂O₂, (B) hydroxyl ion and (C) MDA in the homogenized lung tissue. Data are presented as the mean ± standard error (n=10). *P<0.05 compared with the LPS-induced ALI group; #P<0.05 compared with the vehicle-treated ALI group. MDA, malondialdehyde.

Figure 7

Effect of rhamnazin pretreatment on Nrf2 and Trx1 expression in lung tissues. The mRNA expression levels of (A) Nrf2 and (B) Trx1 are shown. Data are presented as the mean ± standard error (n=10). *P<0.05 compared with the LPS-induced ALI group; #P<0.05 compared with the vehicle-treated ALI group. Nrf2, nuclear factor erythroid-2-related factor 2; Trx1, thioredoxin isoform 1.

Figure 8

Effect of rhamnazin pretreatment on Nrf2 and Trx1 expression in lung tissues. The protein expression levels were determined as (A) western blot analysis (B) Densitometry of western blot was performed to estimate the relative protein concentration. Data are presented as the mean ± standard error (n=10). *P<0.05 compared with the LPS-induced ALI group; #P<0.05 compared with the vehicle-treated ALI group. Nrf2, nuclear factor erythroid-2-related factor 2; Trx1, thioredoxin isoform 1.