Curcumin Ameliorates Ischemia-Induced Limb Injury Through Immunomodulation

Abstract

BACKGROUND The prevalence of peripheral arterial disease (PAD) is increasing worldwide. Currently, there is no effective treatment for PAD. Curcumin is an ingredient of turmeric that has antioxidant, anti-inflammation, and anticancer properties. In the present study we investigated the potential effect of curcumin in protecting against ischemic limb injury. MATERIAL AND METHODS We used an established hindlimb ischemia mouse model in our study. Curcumin was administrated through intraperitoneal (I.P.) injection. Immunohistochemical staining and ELISA assays were performed. Treadmill training was used to evaluate skeletal muscle functions of animals. RESULTS Our experiments using in vivo treadmill training showed that curcumin treatment improved the running capacity of animals after ischemic injury. Histological analysis revealed that curcumin treatment significantly reduced the skeletal muscle damage and fibrosis associated with ischemic injury. In order to determine the cellular and molecular mechanisms underlying curcumin-mediated tissue protection, immunohistochemical staining and ELISA assays were performed. The results showed that curcumin treatment led to less macrophage infiltration and less local inflammatory responses as demonstrated by decreasing TNF-α, IL-1, and IL-6 levels. Further immunofluorescent staining of tissue slides indicated that curcumin treatment inhibited the NF-κB signaling pathway. Finally, curcumin can inhibit NF-kB activation induced by LPS in macrophages. CONCLUSIONS Our study results show that curcumin treatment can ameliorate hindlimb injury following ischemic surgery, which suggests that curcumin could be used for PAD treatment.

Figure 1

Curcumin treatment improves muscle function after ischemic injury. Animals were subjected to treadmill running test at indicated time points. Curcumin treatments significantly improved running ability of the animals after ischemic injury as compared to the PBS control group. Data are presented as mean ±SEM; n=10 per group; * p<0.05.

Figure 2

Curcumin treatment improves muscle morphology of hind limbs induced by ischemia. H&E staining and Masson’s trichrome staining (A) of TA muscle sections. It shows less atrophy and less fibrosis in curcumin treatment skeletal muscle tissues as compared with PBS treatment. The muscle fiber number per mm² of the cross-section of TA muscle (B) and percentage of fibrosis (C) in different groups were quantified and summarized. Data are presented as mean ±SEM; n=10 per group, and a total of 30 sections were analyzed per parameter; * p<0.05. Scale bar: 20 μm.

Figure 3

Curcumin treatment reduces macrophage infiltration and inflammatory responses following ischemic injury to hind limbs. (A) Macrophage infiltration was determined by immunohistochemical staining. The macrophages in ischemic tissue was significantly higher in the PBS-treated group (1399 ± 186 cells) than in the curcumin-treated group (946 ± 130 cells). A total of 27 sections were analyzed. (B) Inflammatory factors in ischemia tissues were determined by ELISA. Curcumin treatment can effectively inhibit levels of pro-inflammatory factors, TNF-α, IL-1β, and IL-6. Data are presented as mean ±SEM; * p<0.05. Scale bar: 20 μm.

Figure 4

Curcumin treatment inhibits activation of NF-κB in ischemic tissue. (A) DAPI staining of nucleus. B. p65 staining in different groups of muscles. Increase in p65 expression was observed in the PBS group and treatment with curcumin decreased p65 expression in the muscle. (B) p65 fluorescent signal was quantified by ImageJ software. The results showed curcumin treatment significantly reduced p65 signaling. A total of 27 sections were analyzed. Data are presented as mean ±SEM; n=6 per group; ** p<0.01.

Figure 5

Curcumin inhibits activation of NF-κB in macrophages. (A) Western blot analysis shows that LPS induced activation of NF-κB signal in macrophages is inhibited by co-treatment of curcumin at different time points (30 min and 60 min). (B) Quantification of Western blot analysis by ImageJ software. Data are presented as mean ±SEM; n=6 per group; ** p<0.01.