Evaluating the efficacy of ethanolic extract of Tapak Liman (Elephantopus scaber L.) leaf in inhibiting pulmonary fibrosis: Mechanisms through anti-fibrotic cytokine promotion

Abstract

Background: Pulmonary fibrosis represents the most prevalent form of idiopathic interstitial pneumonia. The pathogenesis of pulmonary fibrosis using a bleomycin-induced mice model has indicated an imbalanced immune response such as an early massive inflammatory response, followed by fibrosis development. Therapy focused on restraining inflammation is one of the ways to inhibit fibrosis development. Elephantopus scaber ethanolic extract (ESEE) is known to have many beneficial compounds that were proven to possess anti-inflammatory activities, but its prospect in inhibiting pulmonary fibrosis development needs to be investigated.

Aim: This study aimed to evaluate the potency of ESEE treatment in inhibiting fibrosis development in the bleomycin-induced pulmonary fibrosis mice model.

Methods: Healthy male BALB/c mice were divided into seven experimental groups (n = 8): healthy mice (N), vehicle mice (VC), pulmonary fibrosis mice (C-), pulmonary fibrosis received dexamethasone (C+), and pulmonary fibrosis mice received ESEE at a 0.0504 mg/kg body weight (BW) (D1), 0.1008 mg/kg BW (D2), and 0.2016 mg/kg BW (D3). Mice were given ESEE orally and intraperitoneal bleomycin injection daily for 14 days. Mice were then sacrificed on days 7 and 14 and spleens were isolated to determine the production of IL-10, TNF-α, and IFN-γ using flow cytometry.

Results: The results revealed that a remarkable increase of TNF-α was found in the macrophage of pulmonary fibrosis mice model from day 7 to 14. An increase in IFN-γ production was also observed on day 7 and then decreased on day 14. The production of IL-10 was reduced in the fibrosis group at day 7 and continued to increase at day 14. Interestingly, ESEE treatment for 14 days could effectively reduce TNF-α and increase IFN-γ production. ESEE treatment could also maintain a stable production of IL-10 at each time point. ESEE at 0.1004 mg/kg BW (D2) showed the most effective activity in reducing pro-fibrotic cytokine than the dexamethasone group.

Conclusion: Ethanolic extract of ESEE has demonstrated its beneficial prospect in regulating pro-inflammatory and pro-fibrotic cytokine to inhibit fibrosis development.

Keywords: Elephantopus scaber; IFN-γ; IL-10; Pulmonary fibrosis; TNF-α.

Fig. 1.. Flow cytometry result of CD11b⁺TNF-α⁺ cell population at days 7 and 14 for each treatment group. (A) Dot plots demonstrated the CD11b⁺TNF-α⁺ cell population. (B) The representative bar showed the mean relative percentage of the CD11b⁺TNF-α⁺ cell population. Data were shown as mean ± standard deviation (SD). Lowercase marks showed significant differences based on Duncan’s HSD post hoc test (p < 0.05). N: healthy mice group; VC: vehicle control group; F: fibrosis model group with bleomycin; D: positive control group with dexamethasone; D1: ESEE treatment at a dose of 0.0504 mg/kg; D2: ESEE treatment at a dose of 0.1008 mg/kg; E3: ESEE treatment at a dose of 0.2016 mg/kg.

Fig. 2.. Flow cytometry result of CD11b⁺IFN-γ⁺ cell population at days 7 and 14 for each treatment group. (A) Dot plots demonstrated the CD11b⁺IFN-γ⁺ cell population. (B) The representative bar showed the mean relative percentage of the CD11b⁺IFN-γ⁺ cell population. Data were shown as mean ± standard deviation (SD). Lowercase marks showed significant differences based on Duncan’s HSD post hoc test (p < 0.05). N: healthy mice group; VC: vehicle control group; F: fibrosis model group with bleomycin; D: positive control group with dexamethasone; D1: ESEE treatment at a dose of 0.0504 mg/kg; D2: ESEE treatment at a dose of 0.1008 mg/kg; E3: ESEE treatment at a dose of 0.2016 mg/kg.

Fig. 3.. Flow cytometry result of CD4⁺CD25⁺IL-10⁺ cell population at days 7 and 14 for each treatment group. (A) Dot plots demonstrated the CD4⁺CD25⁺IL-10⁺ cell population. (B) The representative bar showed the mean relative percentage of the CD4+CD25+IL-10+ population. Data were shown as mean ± standard deviation (SD). Lowercase marks showed significant differences based on Duncan’s HSD post hoc test (p < 0.05). N: healthy mice group; VC: vehicle control group; F: fibrosis model group with bleomycin; D: positive control group with dexamethasone; D1: ESEE treatment at a dose of 0.0504 mg/kg; D2: ESEE treatment at a dose of 0.1008 mg/kg; E3: ESEE treatment at a dose of 0.2016 mg/kg.