The protective effects of Xuebijing injection on intestinal injuries of mice exposed to irradiation

Abstract

Background: Gastrointestinal (GI) injury is one of the most common side effects of radiotherapy. However, there is no ideal therapy method except for symptomatic treatment in the clinic. Xuebijing (XBJ) is a traditional Chinese medicine, used to treat sepsis by injection. In this study, the protective effects of XBJ on radiation-induced intestinal injury (RIII) and its mechanism were explored.

Methods: The effect of XBJ on survival of irradiated C57BL/6 mice was monitored. Histological changes including the number of crypts and the length of villi were evaluated by H&E. The expression of Lgr5⁺ intestinal stem cells (ISCs), Ki67⁺ cells, villin and lysozymes were examined by immunohistochemistry. The expression of cytokines in the intestinal crypt was detected by RT-PCR. DNA damage and apoptosis rates in the small intestine were also evaluated by immunofluorescence.

Results: In the present study, XBJ improved the survival rate of the mice after 8.0 and 9.0 Gy total body irradiation (TBI). XBJ attenuated structural damage of the small intestine, maintained regenerative ability and promoted proliferation and differentiation of crypt cells, decreased apoptosis rate and reduced DNA damage in the intestine. Elevation of IL-6 and TNF-α was limited, but IL-1, TNF-𝛽 and IL-10 levels were increased in XBJ-treated group after irradiation. The expression of Bax and p53 were decreased after XBJ treatment.

Conclusions: Taken together, XBJ provides a protective effect on RIII by inhibiting inflammation and blocking p53-related apoptosis pathway.

Keywords: Apoptosis; Inflammation; Intestinal injury; Total body irradiation; Xuebijing injection.

FIGURE 1

XBJ improves survival rate of mice after IR. The mice were treated with a 0.4 ml/kg dose XBJ. (A) Kaplan‐Meier survival analysis of mice after 8.0Gy TBI (p = 0.0233, n = 10). (B) The survival rate of mice after 9.0 TBI (p = 0.0004, n = 15). The mortality of XBJ‐treated mice was reduced compared with IR mice. The data show the percentage of surviving mice.

FIGURE 2

XBJ reduces intestinal morphological damage to mice after IR. The mice were sacrificed and the intestinal sections were obtained at 3.5 days after IR. (A) The H&E pictures show the small intestine structure after 9.0 Gy TBI. (B) Bar graph showing the number of crypts after TBI. (C) IHC photographs showing the expression of villi in intestinal sections. (D) Bar graph showing villous height in intestinal sections. The results are represented as mean ± SEM, n = 5 mice per group. ***p < 0.005, **p < 0.01, *p < 0.05. Scale bars: 100 and 50 μm.

FIGURE 3

XBJ maintains the proliferation and differentiation of the ISCs after TBI. The intestinal sections were taken 3.5 days after IR. (A) IHC images showing the expression of Lgr5 in intestinal crypts. (B) Bar graph showing Lgr5⁺ cell counts per five crypts. (C) IHC images showing the expression of Ki67 in control, IR and IR+XBJ mice intestines. (D) Bar graph showing Ki67‐positive cell counts per five crypts. The results are represented as mean ± SEM, n = 5 mice per group. ***p < 0.005, **p < 0.01. Scale bar: 50 μm.

FIGURE 4

XBJ decreases apoptosis in the small intestine after TBI. (A) TUNEL staining for apoptosis analysis. (B) Bar graph showing TUNEL positive cells per field. (C) Representative IF images for caspase‐8 expression (red, caspase‐8; blue, DAPI). (D) Bar graph showing quantitative analysis of caspase8 positive. (E) Representative IF images for the expression of caspase9‐staining (red, caspase9; blue, DAPI). (F) Bar graph showing the statistical results of Caspase9 positive expression. The results are represented as mean ± SEM, n = 5 mice per group. ***p < 0.005, *p < 0.05. Scale bar: 50 and 10 μm.

FIGURE 5

XBJ affects the expression of cytokines in the small intestinal crypt after TBI. The mRNA levels of IL‐6 (A), IL‐10 (B), IL‐1 (C), TNF‐α (D), and TNF‐β (E) in control mice, IR mice and IR+XBJ mice were determined 3.5 days post 9.0 Gy TBI with real‐time PCR. n = 5 mice per group. ***p < 0.005, **p < 0.01, *p < 0.05.

FIGURE 6

XBJ reduces the expression of 𝛾H2AX and p53 in the intestine after TBI. (A) IF photographs showing the expression of 𝛾H2AX in the intestinal sections after IR (red, 𝛾H2AX; blue, DAPI). (B) Bar graph showing quantitative results of 𝛾H2AX positive expression. (C) Representative IF images for p53‐staining in intestinal sections after IR (red, p53; blue, DAPI). (D) Bar graph showing statistical analysis of p53 positive cells. (E) Western blot showing Caspase‐3, Bax and Tubulin expression in the small intestinal crypt. (F) Bone marrow cells were obtained 3 days after 9.0 Gy TBI. 1000 stained cells were counted per mouse, observed with the microscope. The results are represented as mean ± SEM, n = 5 mice per group. ***p < .005, **p < 0.01, *p < 0.05. Scale bar: 10 μm.