巨噬细胞表皮生长因子受体表达对小檗碱抗结直肠癌作用的影响及机制

Effect and mechanism of EGFR expression in macrophages on the anti-cancer effect of berberine on colorectai cancer

目的探讨巨噬细胞表皮生长因子受体（EGFR）表达对结直肠肿瘤生长及对小檗碱（BER）抗结直肠癌作用的影响及机制。方法以巨噬细胞EGFR基因缺陷（Egfrfl/flLysM-Cre）小鼠及巨噬细胞EGFR基因表达（LysM-Cre）小鼠为研究对象，通过氧化偶氮甲烷（AOM）建立结直肠癌模型后，给予BER干预。观察两组小鼠成瘤情况，评价小鼠结直肠长度和肿瘤数量的变化。采用Ki-67免疫组化检测肿瘤细胞增殖，annexin V-异硫氰酸荧光素荧光标记法检测肿瘤细胞凋亡情况，Westernblot检测cleaved-caspase-3蛋白表达水平。结果AOM诱导后，LysM-Cre组和Egfrfl/flLysM-Cre组小鼠结直肠肿瘤数目分别为（10．26±1．43）个和（7．62±1．05）个，差异有统计学意义（P=0．021）；LysM-Cre组和Egfrfl/flLysM-Cre组小鼠结直肠长度分别为（6．04±1．06）cm和（6．39±0．92）cm，差异无统计学意义（P=0．075）。AOM联合BER干预后，LysM-Cre组和Egfrfl/flLysM-Cre组小鼠结直肠肿瘤数目分别为（8．35±1．22）个和（2．66±0．38）个，差异有统计学意义（P=0．006）；LysM-Cre组和Egfrfl/flLysM-Cre组小鼠结直肠长度分别为（7．34±1．16）em和（10．01±1．72）cm，差异有统计学意义（P=0．028）。免疫组化显示，AOM诱导后，LysM-Cre组和Egfrfl/flLysM-Cre组中Ki-67阳性细胞百分比分别为（78．31±3．43）％和（75．85±2．92）％，差异无统计学意义（P=0．282）；AOM联合BER干预后，LysM-Cre组和Egfrfl/flLysM-Cre组中Ki-67阳性细胞百分比分别为（42．43±3．09）％和（29．65±2．47）％，差异有统计学意义（P=0．018）。AOM诱导后，LysM-Cre组和Egfrfl/flLysM-Cre组中annexinV阳性细胞百分比分别为（0．95±0．13）％和（1．13±0．16）％，差异无统计学意义（P=0．175）；AOM联合BER干预后，LysM-Cre组和Egfrfl/flLysM-Cre组中annexinV阳性细胞百分比分别为（32．10±1．97）％和（47．08±2．83）％，差异有统计学意义（P=0．010）。AOM联合BER干预后，LysM-Cre组和Egfrfl/flLysM-Cre组小鼠结直肠癌细胞中cleaved-caspase-3蛋白表达水平分别为119．71±12．87和235．92±19．73，Egfrfl/flLysM-Cre组cleaved-caspas-3蛋白表达水平高于LysM-Cre组（P=0．012）。结论BER对结肠癌细胞生长有抑制作用，敲除巨噬细胞EGFR表达能够进一步增强BER对结直肠癌细胞的抑制作用，其机制可能与抑制结直肠癌细胞增殖和促进细胞凋亡有关。

Objective To investigate the effect and explore its possible mechanisms of epidermal growth factor receptor（EGFR） expression in macrophages on the anti-cancer effect of berberine （BER） on the growth of colorectal cancer. Methods Mice with EGFR gene defects in macrophages （ Egfl/fl a LysM- Cre） and with EGFR gene expression in macrophages （LysM-Cre） （control group） were treated with azoxymethane （AOM） to establish colorectal tumor models. These models were treated with or without berberine （BER） intervention. The number of colorectal tumors and the gut length in the two groups were measured. The proliferation of tumor cells was detected by Ki-67 immunohistochemistry and apoptosis was detected by annexin V-FITC fluorescence labeling. Western blot was used to detect the expression of cleaved- easpase-3 protein. Results After treated with AOM, the coloreetal tumor number was 10.26± 1.43 in the LysM-Cre group and 7.62± 1.05 in the Egfl/fl LysM-Cre group, showing a significant difference （P= 0.021 ）. The gut length was （6.04±1.06） cm in the LysM-Cre group and （6.39±0.92） cm in the gfrfl/flLysM-Cre group, with a non-significant difference between the two groups （P= 0.075 ）. After treated with AOM plus BER intervention, the eolorectal tumor number of the LysM-Cre group was 8.35± 1.22 and that in the Egfl/fl LysM-Cre group was 2.66±0.38, showing a very significant difference between the two groups （P= 0.006）. The gut length of the LysM-Cre group was （7.34± 1.16） cm and that of the Egfl/fl LysM-Cre group was （ 10.01 ± 1.72） cm （P = 0.028 ）. After treated with AOM, the ratio of Ki-67-positive tumor cells in the LysM- Cre group was （78.31±3.43）% and that in the Egfl/fl LysM-Cre group was （75.85±2.92）% （P= 0.282）. After AOM plus BER treatment, the ratio of Ki-67-positive tumor cells in the LysM-Cre group was （42.43± 3.09） % and that in the Egfl/fl LysM-Cre group was significantly lower （ 29.65 ± 2.47 ） % （ P = 0.018 ）. The ratio of annexin V-positive tumor cells was （0.95±0.13）% in the LysM-Cre group, not significantly different from （1.13±0.16）% in the Egfl/fl LysM-Cre group （P=0.175）. After AOM plus BER treatment, the ratio of annexin V-positive tumor cells in the LysM-Cre group was （32.10± 1.97）%, significantly lower than the （47.08 ± 2.83 ） % in the Egfrfvn LysM-Cre group （ P = 0. 010 ）. The level of cleaved-caspase-3 protein expression was 235.92±19.73 in the Egfl/fl LysM-Cre group, significantly higher than the 119.71± 12.87 in the LysM-Cre group （ P = 0. 012）. Conclusions The growth of colorectal cancer ceils in mice can be inhibited by BER treatment, and this anti-cancer effect of BER can be further enhanced by EGFR gene knockout in maerophages. The mechanisms may be related to the inhibition of proliferation and promotion of apoptosis in eolorectal cancer cells.