Berbamine Promotes the Repair of Lower Limb Muscle Damage in Chronic Limb-Threatening Ischemia by Inhibiting Local Inflammation and NF-κB Nuclear Translocation

Abstract

Background/Objectives: Chronic Limb-Threatening Ischemia (CLTI) is a chronic limb ischemic disease caused by vascular lesions, characterized by pain, ulcers, and gangrene, which can be life-threatening in severe cases. The objective of this study is to explore whether Berbamine (BBM) can protect against and repair ischemic muscle tissue in the lower limbs; Methods: Using a mouse hindlimb ischemia (HLI) model, 36 C57BL6 mice were divided into sham, HLI, and HLI+BBM treatment groups. Results: Our findings indicate that BBM can restore motor function and muscle tissue pathology in mice, potentially by inhibiting the nuclear translocation of nuclear factor kappa-B (NF-κB), thereby alleviating tissue inflammation caused by chronic ischemia, reducing muscle cell apoptosis, inhibiting M1 macrophage polarization, and promoting angiogenesis. Conclusions: Our research suggests that BBM has the potential to protect against ischemic damage in lower limb muscle tissue, providing a new approach to the treatment of CLTI.

Keywords: apoptosis; berbamine; inflammation; limb ischemia; nuclear factor kappa-B.

Figure 1

BBM enhances motor function and ameliorates muscle tissue pathology in mice with hindlimb ischemia (HLI). (a) Representative images of gastrocnemius muscles, measured by H&E staining (transverse section). (b) Quantification of the mean cross-sectional area of skeletal muscle HE staining, n = 8 per group. (c,d) Representative images of PAS staining and quantification of gastrocnemius muscles, transverse section, n = 8 per group. (e,f) Representative images of oil red O staining and quantification of gastrocnemius muscles, transverse section, n = 8 per group. (g,h) Representative images of Masson staining and quantification of gastrocnemius muscles, transverse section, n = 8 per group. (i) The endurance running distance of mice in each group over a fixed time, n = 8 per group. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

BBM promotes angiogenesis and skeletal muscle regeneration in the ischemic hind limbs of mice. (a,b) Representative immunohistochemistry images and quantification of CD31 in gastrocnemius muscles transverse sections, n = 8 per group. (c) The mRNA expression of VEGFA in gastrocnemius muscles examined by qPCR, n = 3 per group. (d,e) Representative immunofluorescence images and quantification of Edu in HUVECs, n = 3 per group. (f,g) Representative images and quantification of relative tube length and number of junctions in angiogenesis test, n = 3 per group. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

BBM inhibits M1 polarization of macrophages and inflammation in the ischemic hind limbs of mice. (a) The mRNA expression of iNOS, IL-6, IL-10, and Arg1 in gastrocnemius muscles examined by qPCR, n = 3 per group. (b,c) Representative immunofluorescence images and quantification of CD86 in gastrocnemius muscles transverse sections, n = 8 per group. (d,e) Representative immunofluorescence images and quantification of CD163 in gastrocnemius muscles transverse sections, n = 8 per group. (f) mRNA expression level of IL-1β and TNF-α in the gastrocnemius muscles of mice, detected by qPCR, n = 3 per group. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns: not significant.

Figure 4

BBM inhibits apoptosis induced by hind limb ischemia in mice. (a,b) Representative immunofluorescence images and quantification of Tunel staining in gastrocnemius muscles transverse sections, n = 8 per group. (c,d) Representative immunoblot bands and quantitative analysis of cleaved-Caspase-3 and cleaved-Parp1 in gastrocnemius muscles, n = 3 per group. Data are expressed as mean ± SEM. **** p < 0.0001.

Figure 5

Berbamine inhibits NF-κB nuclear translocation. (a,b) nuclear translocation was determined by immunofluorescence staining for p65 (red) and DAPI staining for DNA (blue), n = 8 per group. (c,d) Nuclear p65 protein expression in gastrocnemius muscles was analyzed by western blot, n = 3 per group. Data are expressed as mean ± SEM. **** p < 0.0001.