益肾固骨方基于Wnt5a/β-catenin信号通路调控炎性因子治疗肺腺癌骨转移实验研究

Experimental study of YSGG prescription based on Wnt5a/β-catenin signaling pathway regulating inflammatory factors in the treatment of lung adenocarcinoma bone metastasis

目的建立肺腺癌骨转移小鼠模型,探讨益肾固骨方通过Wnt5a/β-catenin通路对肺腺癌骨转移小鼠股骨处破骨细胞的影响。方法70只C57BL/6雄性小鼠,采用随机区组法随机分组,将小鼠随机分配至7个组(每组10只)。从中任意选取1组小鼠,注射PBS至左后肢股骨,作为假手术组;接种Lewis细胞悬液至其余60只小鼠左后肢股骨。假手术组和模型对照组小鼠用生理盐水灌胃,中药组(低)、中药组(中)、中药组(高)分别灌胃3、12、24g/kg剂量的益肾固骨方,西药组腹腔注射唑来膦酸(50μg/kg),中西联合用药组灌胃低剂量的益肾固骨方并腹腔注射唑来膦酸(50μg/kg),治疗21d。抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色法200倍光镜下观察小鼠左后肢股骨处破骨细胞数量,RT-PCR法检测Wnt5a、β-catenin、Gsk-3β、p-Gsk-3β、Dkk1、Tnf-α、Il-17 mRNA的表达,免疫组织化学法检测WNT5A、β-catenin、GSK-3β、p-GSK-3β、DKK1、TNF-α、IL-17蛋白的表达。采用单因素或双因素方差分析对数据进行统计分析。结果(1)破骨细胞数量结果表明,7组间均值差异有统计学意义,F=13.113,P<0.001,两两多重比较显示,除了中药组(中),模型对照组与其余各组比较,均P<0.001。模型组破骨细胞数量最高(73±13.61),而中药组低剂量(41±8.93)、高剂量(41±17.63)、联合用药组(51±7.95)破骨细胞数量接近假手术组(45±13.06)。(2)与假手术组(2.30±1.27,1.37±0.45)相比,模型组(18.71±2.29,11.50±5.17)的β-catenin mRNA和Gsk-3βmRNA表达升高,差异有统计学意义,P<0.001;与模型组(18.71±2.29,11.50±5.17)比较,西药组(1.62±0.19,0.26±0.10)、联合用药组(1.79±0.28,0.46±0.16)的β-catenin和Gsk-3βmRNA表达均降低,差异均有统计学意义,均P<0.001。(3)与假手术组(0.92±0.36,1.86±1.07,0.95±0.23)比较,模型对照组(0.21±0.13,0.16±0.03,0.54±0.30)的Wnt5a、β-catenin和DKK1蛋白染色阳性面积降低,差异均有统计学意义,均P<0.01;与假手术组(3.34±2.47,0.95±0.02)比较,模型对照组(14.63±2.11,6.55±2.57)的GSK-3β、p-GSK-3β蛋白染色阳性面积升高,差异均有统计学意义,均P<0.01。与模型对照组(0.92±0.36)相比,西药组(2.57±0.82)和联合用药组(2.84±0.92)的Wnt5a染色阳性面积升高,差异均有统计学意义,均P<0.01。与模型对照组(0.16±0.03)相比,中药组中剂量(0.37±0.29)和联合用药组(0.60±0.21)的β-catenin染色阳性面积升高,差异均有统计学意义(P<0.05,P<0.01)。与模型对照组(14.63±2.11)相比,假手术组(3.34±2.47)、中药组低剂量(1.01±0.59)、中剂量(3.94±1.44)的GSK-3β蛋白染色阳性面积降低,差异均有统计学意义,均P<0.001。与模型对照组(14.63±2.11,6.55±2.57)相比,中药组高剂量(2.71±1.49,4.50±0.39)、西药组(0.76±0.38,0.92±0.61)、联合用药组(3.66±1.12,0.54±0.24)的GSK-3β、p-GSK-3β蛋白染色阳性面积降低,差异均有统计学意义,均P<0.001。与模型对照组(0.54±0.30)相比,中药组中剂量(0.76±0.08)、西药组(0.72±0.07)的DKK1染色阳性面积升高,差异有统计学意义,P<0.01,P<0.05。(4)与模型对照组(0.17±0.11)相比,西药组(3.52±0.87)和联合用药组(3.57±2.13)的Il-17 mRNA表达增加,差异均有统计学意义,均P<0.001。与假手术组(1.00±0.27)相比,模型对照组(3.59±1.18)的Tnf-αmRNA表达增加,差异有统计学意义,P<0.01。与模型对照组(3.59±1.18)相比,西药组(0.28±0.18)和联合用药组(0.29±0.16)的Tnf-αmRNA表达降低,差异均有统计学意义,均P<0.001。(5)与模型对照组(2.83±0.64)相比,中药组低剂量(3.91±0.14)、联合用药组(3.92±2.07)的IL-17蛋白的染色阳性面积升高,差异均有统计学意义,均P<0.01。与假手术组(3.92±1.64)相比,模型对照组(7.84±3.38)的TNF-α蛋白染色阳性面积升高,差异有统计学意义,P<0.05。与模型对照组(7.84±3.38)相比,中药组高剂量(3.26±1.24)、西药组(0.78±0.12)和联合用药组(0.69±0.13)的TNF-α染色阳性面积降低,差异均有统计学意义,均P<0.01。结论益肾固骨方可能通过Wnt5a/β-catenin信号通路及下游分子调控炎性因子治疗肺腺癌骨转移。

OBJECTIVE To establish a mouse model of lung adenocarcinoma bone metastasis to investigate the effect of YSGG prescription on femur osteoclasts of such mice with lung adenocarcinoma bone metastasis via Wnt5 a/β-catenin pathway.METHODS Seventy C57 BL/6 male mice were randomly divided into seven groups based on their body weight(10 mice per group).One group of mice was randomly selected and injected with PBS into the left hind limb femur as the sham operation group.Lewis cell suspension was inoculated to the remaining 60 mice’s left hind limb femur.Gastric lavage was performed on sham operation group and model control group with saline water,while TCM group(low),TCM group(middle)and TCM group(high)were given YSGG prescription at doses of 3,12,24 g/kg respectively;western medicine group was injected with intraperitoneal to phosphonic acid(50μg/kg),and the combination group was lavaged with low doses of intraperitoneal injection of solid bone side kidney and thiazole to phosphonic acid(50μg/kg),with treatment period of 21 days.The number of osteoclasts in the femur of the left hind leg of mice was observed under 200 times light microscope by tartrate-resistant acid phosphatase(TRAP)staining.The expressions of Wnt5 a,β-catenin,Gsk-3β,Dkk1,Tnf-αand Il-17 mRNA were examined by RT-PCR.The expressions of WNT5 A,β-catenin,GSK-3β,p-GSK-3β,DKK1,TNF-αand IL-17 protein were examined by immunohistochemical method.Statistical methods were single factor or2 factor analysis of variance.RESULTS(1)The number of osteoclast showed that the average difference between7 groups had statistical significance(F=13.113,P<0.001),pairwise multiple comparisons demonstrated that the model control groups was compared with other groups excluding TCM group(middle),all P<0.001.The highest number of osteoclast was on model control group(73±13.61),while the numbers of osteoclast in low dose TCM group(41±8.93),high does TCM group(41±17.63)and the combination group(51±7.95)were closed to that in sham operation group(45±13.06).(2)Compared with sham operation group(2.30±1.27,1.37±0.45),the expression ofβ-catenin mRNA and Gsk-3βmRNA in the model control group(18.71±2.29,11.50±5.17)was increased,with statistically significant difference(P<0.001).Compared with the model control group(18.71±2.29,11.50±5.17),the expressions ofβ-catenin mRNA and Gsk-3βmRNA in the western medicine group(1.62±0.19,0.26±0.10)and the combination group(1.79±0.28,0.46±0.16)were all decreased,and the differences were statistically significant(all P<0.001).(3)Compared with sham operation group(0.92±0.36,1.86±1.07,0.95±0.23),the positive staining area of Wnt5 a,β-catenin and DKK1 protein in model control group(0.21±0.13,0.16±0.03,0.54±0.30)were decreased,and the differences were statistically significant,all P<0.01;Compared with sham operation group(3.34±2.47,0.95±0.02),the positive staining areas of GSK-3βand p-GSK-3βprotein in model control group(14.63±2.11,6.55±2.57)were increased,and the difference was statistically significant,all P<0.01.Compared with the model control group(0.92±0.36),the staining positive area of WNT5 Aprotein in the western medicine group(2.57±0.82)and the combination group(2.84±0.92)was increased,and the differences were statistically significant,all P<0.01.Compared with the model control group(0.16±0.03),the positive staining area ofβ-catenin in the middle dose TCM group(0.37±0.29)and the combination group(0.60±0.21)was increased,and the difference was statistically significant(P<0.05,P<0.01).Compared with the model control group(14.63±2.11),the staining positive area of GSK-3βprotein in the sham operation group(3.34±2.47),the low dose TCM group(1.01±0.59),and middle dose TCM group(3.94±1.44)were lower,and the difference was statistically significant,all P<0.001.Compared with the model control group(14.63±2.11,6.55±2.57),the staining positive area of GSK-3βand p-GSK-3βprotein in the high dose TCM group(2.71±1.49,4.50±0.39),the western medicine group(0.76±0.38,0.92±0.61),and the combined treatment group(3.66±1.12,0.54±0.24)was decreased,and the difference was statistically significant,all P<0.001.Compared with the model control group(0.54±0.30),the staining positive area of DKK1 protein in the middle dose TCM group(0.76±0.08)and western medicine group(0.72±0.07)was increased,and the difference was statistically significant(P<0.01,P<0.05).(4)Compared with the model control group(0.17±0.11),the Il-17 mRNA expression of the western medicine group(3.52±0.87)and the combination group(3.57±2.13)was increased,and the difference was statistically significant,all P<0.001.Compared with the sham operation group(1.00±0.27),Tnf-αmRNA expression of the model control group(3.59±1.18)was increased,and the difference was statistically significant(P<0.01).Compared with the model control group(3.59±1.18),Tnf-αmRNA expression of the western medicine group(0.28±0.18)and the combination group(0.29±0.16)was decreased,and the difference was statistically significant,all P<0.001.(5)Compared with the model control group(2.83±0.64),the staining positive area of IL-17 protein in the low dose TCM group(3.91±0.14)and the combination group(3.92±2.07)was increased,and the difference was statistically significant,all P<0.01.Compared with the sham operation group(3.92±1.64),the staining positive area of TNF-αprotein in the model control group(7.84±3.38)was increased,and the difference was statistically significant,P<0.05.Compared with the model control group(7.84±3.38),the staining positive area of TNF-αin the high dose TCM group(3.26±1.24),the western medicine group(0.78±0.12)and the combination group(0.69±0.13)were decreased,and the difference was statistically significant,all P<0.01.CONCLUSION YSGG prescription has the possibility of treating lung adenocarcinoma bone metastasis by regulating inflammatory factors via Wnt5 a/β-catenin signaling pathway and its downstream molecules.