Dl-3-n-Butylphthalide (NBP) Mitigates Muscular Injury Induced by Limb Ischemia/Reperfusion in Mice through the HMGB1/TLR4/NF- κ B Pathway

Abstract

Objective: Limb ischemia/reperfusion (I/R) injury is a clinical syndrome associated with severe damages to skeletal muscles and other fatal outcomes. Oxidative stress and inflammatory response play vital roles in the development of limb I/R injury. Existing evidence further indicates that Dl-3-n-butylphthalide (NBP) has anti-inflammatory and antioxidative properties. However, whether NBP can protect skeletal muscles from limb I/R injury and the mechanism in mediating the action of NBP treatment still remain to be investigated, which are the focuses of the current study.

Methods: The model of limb I/R injury was established and H&E staining was adopted to assess the pathological changes in skeletal muscles following limb I/R injury. Additionally, the W/D ratio of muscle tissue was also measured. ELISA and biochemical tests were carried out to measure the levels of inflammatory cytokines and oxidative stress in mouse models of limb I/R injury. Moreover, the levels of the HMGB1/TLR4/NF-κB pathway-related proteins were also determined using immunohistochemistry and immunoblotting.

Results: It was established that NBP treatment alleviated I/R-induced pathological changes in muscular tissue of mice, accompanied by lower W/D ratio of skeletal muscular tissue. Meanwhile, the limb I/R-induced inflammation and oxidative stress in skeletal muscles of mice were also inhibited by NBP. Mechanistic study indicated that the alleviatory effect of NBP was ascribed to inactivation of the HMGB1/TLR4/NF-κB pathway.

Conclusions: Our findings highlighted the potential of NBP as a novel strategy for limb I/R-driven muscle tissue damages by suppressing inflammatory response and oxidative stress via the HMGB1/TLR4/NF-κB pathway.

Figure 1

Effect of NBP on pathological change and wet weight/dry weight ratio of skeletal muscle in I/R-injured mice. (a) H&E staining was used to observe the degree of inflammation of muscular tissue in each group of mice (bar = 50 μm); (b) The pathological score in each group of mice was quantified; (c) The wet weight/dry weight ratio of skeletal muscle was calculated to assess the extent of edema in different groups of mice. (d) The level of LDH was measured in muscular homogenates of each group of mice; (e) The level of CK-MB was measured in muscular homogenates of each group of mice. n = 10 for each group. ^∗p < 0.05 and ^∗∗p < 0.01.

Figure 2

Effect of NBP on inflammatory responses of skeletal muscles in I/R-injured mice. (a) (b) and (c) ELISA was conducted to determine the levels of IL-1β (a), TNF-α (b), and IL-6 (c) in the homogenates of muscular tissues of each group of mice; n = 10 for each group. ^∗p < 0.05 and ^∗∗p < 0.01.

Figure 3

Effect of NBP on oxidative stress of skeletal muscles in I/R-injured mice. (a) The production of MDA was measured in muscular homogenates of each group of mice; (b) The activity of SOD was measured in muscular homogenates of each group of mice. n = 10 for each group. ^∗p < 0.05 and ^∗∗p < 0.01.

Figure 4

Effect of NBP on activation of the HMGB1/TLR4/NF-κB pathway in I/R-injured mice. (a) Immunohistochemistry was conducted to assess the quantity of HMGB1 and TLR4 positive cells in each group of mice (bar = 50 μm); (b) Immunoblotting was used to examine the protein expressions of HMGB1, TLR4, Myd88, and the extent of NF-κB p65 phosphorylation in the muscular homogenates in each group of mice. n = 3 ^∗p < 0.05and ^∗∗p < 0.01.

Figure 5

In summary, this study dissected out the effect of NBP on the limb I/R injury-driven skeletal muscle damages, and our findings suggested that NBP could protect limbs against I/R injury by inhibiting inflammation responses and oxidative stress via the HMGB1/TLR4/NF-κB pathway.