Ruan Jian Qing Mai Recipe Inhibits the Inflammatory Response in Acute Lower Limb Ischemic Mice through the JAK2/STAT3 Pathway

Abstract

Ruan jian qing mai recipe (RJQM) is an empirical prescription for treating arteriosclerosis obliterans (ASO). However, the mechanism of RJQM recipe-mediated ASO attenuation has not yet been elucidated. Therefore, this study aimed to explore the mechanism by which the RJQM recipe relieves ASO in a mouse model of lower limb ischemia, which was established by ligating and breaking the femoral artery of the left lower limb. The surgical groups were divided into the ischemic group, beraprost sodium group, low-dose RJQM group, medium-dose RJQM group, and high-dose RJQM group. Normal mice were set as the control group. The blood flow of the lower limb was examined on days 7 and 14. At the end of animal procedures, blood samples were collected, and the rectus femoris of the left lower limb were harvested. Results revealed that mice in the ischemic group demonstrated low blood flow. Additionally, hematoxylin and eosin, and Masson staining results showed that inflammation of the rectus femoris was obvious in the ischemia group, and the level of fibrosis was increased. Blood flow was recovered in all treatment groups compared to the ischemic group, and the inflammatory infiltration and fibrosis of the rectus femoris were relieved after RJQM treatment. The serum levels of interleukin (IL)-17A and IL-21 were decreased, and the expression of JAK2/STAT3 proteins was inhibited in all RJQM treatment groups compared to the ischemia group. Furthermore, the improvement of IL-17A, IL-21, and rectus femoris fibrosis was more obvious with increasing treatment time. In conclusion, RJQM can effectively alleviate ASO and promote the recovery of lower limb blood flow by regulating the JAK2/STAT3 signaling pathway to reduce the inflammatory response.

Figure 1

Changes in hindlimb blood flow in mice. (a) Representative blood flow images of hindlimbs of mice on days 0, 7, and 14. (b) A comparison of blood flow values of the left and right hindlimbs between the groups on the same day of the operation. (c) A comparison of blood flow values of the left and right hindlimbs between the groups on the 7^th day. (d) A comparison of blood flow values of the left and right hindlimbs between the groups on the 14^th day. Scale bar = 2 cm; CON, control group; ISC, ischemic group; BPS, sodium beraprost; L low dose of RJQM; M medium dose of RJQM; and H high dose of RJQM. ^∗P < 0.05, ^∗∗∗∗P < 0.0001. The data are expressed as ± standard error of the mean (SEM) (n = 10 in each group).

Figure 2

The inflammatory manifestations were evaluated with H&E staining. The representative images of the rectus femoris of the left hindlimb of the experimental group and the control group were treated for 7 and 14 days. Inflammatory cell infiltration (↙); Muscle fiber swelling (^∗). Magnifications: ×400, Scale bar = 60 μm. CON, control group; ISC, ischemic group; BPS, sodium beraprost; L low dose of RJQM; M medium dose of RJQM; and H high dose of RJQM.

Figure 3

Evaluation of fibrosis according to Masson staining. (a) Representative images of rectus femoris of the left hindlimb in the experimental group and the control group on the 7^th and 14^th days. (b) Quantification of the percentage area of collagen-positive cells (blue) in each image. Magnifications: ×200, Scale bar = 100 μm. CON, control group; ISC, ischemic group; BPS, sodium beraprost; L low dose of RJQM; M medium dose of RJQM; and H high dose of RJQM. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. The data expressed as ± standard error of the mean (SEM) (n = 3 in each group).

Figure 4

Serum levels of IL-17A and IL-21 in mice. (a) and (b) The levels of IL-17A and IL-21 in the serum of mice in each group were observed after seven days. (c) and (d) the levels of IL-17A and IL-21 in the serum of mice after 14 days. CON, control group; ISC, ischemic group; BPS, sodium beraprost; L low dose of RJQM; M medium dose of RJQM; and H high dose of RJQM. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, ^∗∗∗∗P < 0.0001. The data are expressed as ± standard error of the mean (SEM) (n = 6 for each group).

Figure 5

Expression of JAK2, STAT3, and IL-17A in the rectus femoris. (a and c) Representative western blots of JAK2, STAT3, and IL-17A protein levels in the rectus femoris after 7 and 14 days, with β-tubulin as control. (b and d) An average gray value of JAK2, STAT3, and IL-17A protein levels in the rectus femoris on the 7^th and 14th day. CON, control group; ISC, ischemic group; BPS, sodium beraprost; L low dose of RJQM; M medium dose of RJQM; and H high dose of RJQM. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. The data are expressed as ± standard error of the means (SEM) (n = 3 for each group).