Tinospora cordifolia protects against inflammation associated anemia by modulating inflammatory cytokines and hepcidin expression in male Wistar rats

Abstract

Systemic iron homeostasis dysregulation is primarily associated with inflammation- associated anemia (AI) due to hepcidin up-regulation. Tinospora cordifolia (TC) has shown remarkable anti-inflammatory properties and has been found useful in the treatment of inflammatory disorders. However, the effects and mechanisms of TC on AI have not been studied yet. We conducted in vivo and in vitro studies to evaluate the effect of TC on AI. HPLC studies were also carried out to find out active constituents in TC extract. Model system exhibiting AI was developed by repeated injections of HKBA in Wistar rats. TC treated groups showed significantly higher levels of Hb and RBC count compared to the inflammatory control group. TC treatment showed reduction in the expression of the HAMP (hepcidin) gene in the rat liver. TC extract also inhibited gene expression of inflammatory cytokines (TNF-α, IL-1β) and decreased NO production in RAW 264.7 cells. The HPLC analysis revealed the presence of tinosporaside, which could have synergistically contributed to the above findings. Overall results indicate that TC therapy was able to maintain circulating iron through reduction of inflammatory cytokines and expression of hepcidin in rats.

Figure 1

Effect on Hb levels (A–D) and RBC count (E–H) in rats treated/untreated with TC. IC: Inflammatory control, 100: 100 mg/kg, 200: 200 mg/kg and 400: 400 mg/kg of body weight of TC. Number of animals per group = 5. Results are shown as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 against the IC group and ^##p < 0.01 and ^###p < 0.001 against control.

Figure 2

Changes in serum iron levels in rats treated groups. IC: Inflammatory control, 100: 100 mg/kg, 200: 200 mg/kg, and 400: 400 mg/kg of body weight of TC. Number of animals per group = 5. Results are shown as mean ± SEM. *p < 0.05, against the IC group and ^##p < 0.01 and ^###p < 0.001 against control.

Figure 3

Effects of TC on liver and spleen iron levels. IC: Inflammatory control, 100: 100 mg/kg, 200: 200 mg/kg, and 400: 400 mg/kg of body weight of TC. Number of animals per group = 5. Results are shown as mean ± SEM. *p < 0.05 and **p < 0.01 against the IC group and ^##p < 0.01 and ^###p < 0.001 against control.

Figure 4

TC protects infection-induced liver damage. (A–F) Slides were observed under a light microscope at 40× magnification. In the figure white arrow shows the dilation of the bile duct, black arrow shows the infiltration of inflammatory cells, dashed arrow shows the necrosis, and red arrow shows tissue swelling and loss of tissue architecture. NC: Normal control, IC: Inflammatory control, TC 100: 100 mg/kg, TC 200: 200 mg/kg, and TC 400: 400 mg/kg of body weight of TC aqueous extract. Number of animals per group = 5. Scale bar = 50 µm.

Figure 5

Protective effect of TC against infection-induced splenomegaly. (A–F) In the figure, white arrow shows depletion of white pulp, black arrow shows cellular rapture and structural changes, and red arrow shows necrotic white patches. NC: Normal control, IC: Inflammatory control, TC 100: 100 mg/kg, TC 200: 200 mg/kg, and TC 400: 400 mg/kg of body weight of TC aqueous extract. Number of animals per group = 5. Scale bar = 50 µm.

Figure 6

Effect of TC on hepatic expression of HAMP (A) TLR-4 (B) TNF-α (C) and COX-2 (D) genes. IC: Inflammatory control, 100: 100 mg/kg, 200: 200 mg/kg, and 400: 400 mg/kg of body weight of TC aqueous extract. Number of animals per group = 5. Results are shown as mean ± SEM. Statistical analyses were carried out by using one-way ANOVA and Bonferroni post-hoc test with *p < 0.05, **p < 0.01 and ***p < 0.001 against the IC group and ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 against control.

Figure 7

Effects of TC extract on NO production in RAW 264.7 cells. Data are expressed as the mean ± SEM of at least three independent experiments. Statistical analyses were carried out by using one-way ANOVA and Bonferroni post-hoc test with ***p < 0.001 against the LPS-stimulated positive control.

Figure 8

Effects of TC extract on LPS-induced mRNA expression of HAMP (A) TNF-α (B) IL-1β (C) genes in RAW264.7 cells. Results are in Means ± SEM are shown. IC: Cells treated with LPS (1 µg/mL). **p < 0.01 and ***p < 0.001 against the LPS-stimulated positive control.

Figure 9

The chromatogram of TC extract showing  the presence of tinosporaside (A). The chromatogram of pure  tinosporaside (B).

Figure 10

Effects of tinosporaside on NO production in RAW 264.7 cells. Data are expressed as the mean ± SEM of at least three independent experiments. Statistical analyses were carried out by using one-way ANOVA and Bonferroni post-hoc test with *p < 0.05, ***p < 0.001 against the LPS-stimulated positive control.

Figure 11

Experimental timeline diagram indicating various stages involved in this study.