Our previous research demonstrated that the Wenxia Changfu Formula(WCF),as a neoadjuvant therapy,inhibits M2 macrophage infiltration in the tumor microenvironment and prevents lung cancer metastasis.Given tumor-associ...Our previous research demonstrated that the Wenxia Changfu Formula(WCF),as a neoadjuvant therapy,inhibits M2 macrophage infiltration in the tumor microenvironment and prevents lung cancer metastasis.Given tumor-associated macrophages(TAMs)in epithelial-mesenchymal transition(EMT),this study investigated whether WCF impedes lung cancer metastasis by attenuating TAM-induced EMT in non-small cell lung cancer(NSCLC)cells.Utilizing a co-culture model treated with or without WCF,we observed that WCF downregulated cluster of differentiation 163(CD163)expression in macrophages,reduced CCL18 levels in the conditioned medium,and inhibited the growth,invasion,and EMT of NSCLC cells induced by macrophage co-culture.Manipulation of CCL18 levels and Src overexpression in NSCLC cells revealed that WCF’s effects are mediated through CCL18 and Src signaling.In vivo,WCF inhibited recombinant CCL18(rCCL18)-induced tumor metastasis in nude mice by blocking Src signaling.These findings indicate that WCF inhibits NSCLC metastasis by impeding TAM-induced EMT via antagonistic modulation of CCL18,providing evidence for its potential development and clinical application in NSCLC patients.展开更多
Objective： To explore the apoptosis mechanism of Wenxia Changfu Formula （温下肠腑方, WCF） in reversing drug resistance of lung cancer in vivo. Metbods： Thirty model mice were randomly assigned to three groups： co...Objective： To explore the apoptosis mechanism of Wenxia Changfu Formula （温下肠腑方, WCF） in reversing drug resistance of lung cancer in vivo. Metbods： Thirty model mice were randomly assigned to three groups： control group, cisplatin （CDDP） group, and WCF group. A transplanted tumor model of lung adenocarcinoma was established in all groups. Mice in the WCF group received intragastric administration of WCF （0.2 mL/10 g body weight） everyday in addition to CDDP intraperitoneally （5 mg/kg body weight） twice a week. The mice in the CDDP group received CDDP intraperitoneally （5 mg/kg body weight） twice a week, while the control group received normal saline intraperitoneally （0.2 mL/10 g body weight） everyday. The weight of the nude mice and respective tumors, tumor volume and tumor-inhibiting rate were measured. Electron microscopy was used to observe the existence of apoptosis body. Apoptosis index （AI） was detected by TdT-mediated dUTP nick end labeling staining. The expression of Fas and FasL mRNA was investigated by reverse transcription polymerase chain reaction, while immunohistochemistry was applied to detect the protein expression of Fas and FasL, caspase-3 and caspase-activated DNase （CAD）, respectively. Results： Compared with CDDP group and control group, WCF could significantly reduce the tumor volume from the 19th day and alleviate the tumor weight （P〈0.05）, and the apoptosis body was found in tumor cells in the WCF group. WCF could also enhance the level of AI, up-regulate the expression of caspase apoptosis pathway related protein caspase-3 and CAD, as well as the expression of Fas, FasL mRNA and protein （P〈0.05）. Conclusion： WCF could improve the sensitivity of tumor cells to CDDP and reverse the drug resistance by inducinq the apoptosis.展开更多
目的:探讨温下方对巨噬细胞极化调控机制及M2型巨噬细胞(M2-TAMs)对A549细胞侵袭转移的影响。方法:动物实验:BALB/c裸鼠随机分为模型组和温下方组,灌胃干预。活体成像分析光子强度;观察肺部病理变化;识别CD68/CD163双标M2-TAMs。细胞实...目的:探讨温下方对巨噬细胞极化调控机制及M2型巨噬细胞(M2-TAMs)对A549细胞侵袭转移的影响。方法:动物实验:BALB/c裸鼠随机分为模型组和温下方组,灌胃干预。活体成像分析光子强度;观察肺部病理变化;识别CD68/CD163双标M2-TAMs。细胞实验:佛波酯(PMA)及白介素-4(IL-4)诱导M2-TAMs;流式细胞术检测M2-TAMs标志蛋白CD163表达;PCR检测温下方对M2-TAMs相关基因mRNA表达的影响;划痕和Transwell检测温下方对M2-TAMs促A549细胞侵袭转移的影响;Western blot和PCR检测非受体型酪氨酸蛋白激酶3(J AK3)、信号转导及转录激活因子6(ST AT6)和过氧化物酶增殖物激活受体γ(PP A Rγ)蛋白及mRNA表达。结果:动物实验显示,与模型组比较,温下方在第28天~35天有效降低转移灶光子强度(P<0.05,P<0.01);温下方有效减轻肺部病理损伤;温下方降低转移瘤中M2-TAMs特异性表型CD68和CD163双阳表达。细胞实验显示,成功诱导的M2-TAMs高表达CD163;与M2组比较,温下方显著抑制M2-TAMs标志物Arg-1、Fizz-1、C D206 m R NA表达(P<0.01);与M2组比较,M2+WC F组显著降低M2-T A M s促A549细胞的迁移和侵袭能力(P<0.01);与M2组相比较,温下方有效降低p-JAK3、p-STAT6和PPARγ蛋白及JAK3、STAT6、PPARγmRNA表达(P<0.05,P<0.01)。结论:温下方可能通过调控JAK3/STAT6/PPARγ信号通路,抑制巨噬细胞M2极化,降低A549细胞侵袭转移。展开更多
Objective： To observe the effect of the combination of Wenxia Changfu Formula （温下肠腑方, WCF） with cisplatin （CDDP） on inhibiting non-small cell lung cancer （NSCLC） in vitro and in vivo and explore its mechan...Objective： To observe the effect of the combination of Wenxia Changfu Formula （温下肠腑方, WCF） with cisplatin （CDDP） on inhibiting non-small cell lung cancer （NSCLC） in vitro and in vivo and explore its mechanism from its effect on cell cycle. Methods： In vitro, WCF-containing serum was prepared and the rhubarb bl, emodin, and aconitine were detected qualitatively by high-performance liquid chromatogram （HPLC）. A549 cell lines were treated with blank control （dimethyl sulfoxide）, normal serum, normal serum with CDDP （1.25, 2.5, and 5.0 μg/mL, respectively）, WCF-containing serum plus different doses of CDDP （1.25, 2.5, and 5.0 μ g/mL, respectively）. The inhibitory effect was detected by 3-（4,5）-dimethylthiazo（-z- yl）-3,5-diphenylterazolium bromide （MI-I）. The cell cycle was detected by flow cytometry. The protein and mRNA expressions of cyciin D1, proliferating cell nuclear antigen （PCNA）, retinoblastoma （Rb）, and p16 were observed with immunofluorescence and RT-PCR, respectively. In vivo, nude mice xenograft model was established and grouped into the control, CDDP, WCF, and combination groups. The combination＇s inhibition of tumor growth and influence on the weight, spleen, and thymus gland were observed. Results： The inhibitory rate of the combination against A549 cell lines excelled the CDDP alone significantly （P〈0.05）; the combination showed a synergism inhibitory effect （Q=1.19）. Compared with the monotherapy, the combination increased the cell percentage in G0/G1 phase and decreased the cell percentage in S phase significantly （P〈0.05）; the protein and mRNA expressions of cyclin D1, PCNA, and Rb were significantly reduced; the protein and mRNA expressions of p16 were significantly enhanced. Compared with the monotherapy, the combination inhibited the tumor growth significantly in vivo and reduced the weight of tumor （P〈0.05）; compared with the CDDP group, the spleen and thymus gland index of the combination group were enhanced significantly （P〈0.05）. Conclusions： The combination of WCF with CDDP significantly inhibited the A549 cell lines proliferation in vitro and the growth of the tumor in vivo; it inhibited effectively the atrophy of the immune organ caused by chemotherapy. The combination inhibited overproliferation of A549 cell lines by arresting the Go/G1 phase of cell cycle and affecting the protein and mRNA expressions of cell cycle-related proteins, cyclin D1, etc.展开更多
基金supported by the National Natural Science Foundation of China(No.82274406,81774198)Zhejiang Provincial Natural Science Foundation of China(No.LZ24H270001)the Postgraduate Scientific Research Fund of Zhejiang Chinese Medical University(No.2022YKJ14).
文摘Our previous research demonstrated that the Wenxia Changfu Formula(WCF),as a neoadjuvant therapy,inhibits M2 macrophage infiltration in the tumor microenvironment and prevents lung cancer metastasis.Given tumor-associated macrophages(TAMs)in epithelial-mesenchymal transition(EMT),this study investigated whether WCF impedes lung cancer metastasis by attenuating TAM-induced EMT in non-small cell lung cancer(NSCLC)cells.Utilizing a co-culture model treated with or without WCF,we observed that WCF downregulated cluster of differentiation 163(CD163)expression in macrophages,reduced CCL18 levels in the conditioned medium,and inhibited the growth,invasion,and EMT of NSCLC cells induced by macrophage co-culture.Manipulation of CCL18 levels and Src overexpression in NSCLC cells revealed that WCF’s effects are mediated through CCL18 and Src signaling.In vivo,WCF inhibited recombinant CCL18(rCCL18)-induced tumor metastasis in nude mice by blocking Src signaling.These findings indicate that WCF inhibits NSCLC metastasis by impeding TAM-induced EMT via antagonistic modulation of CCL18,providing evidence for its potential development and clinical application in NSCLC patients.
基金Supported by the National Natural Science Foundation of China(No.81072832,81273634)the Opening Project of Shanghai Key Laboratory of Complex Prescription(No.11DZ2272300)National Science and Technology Major Projects for"Major New Drugs Innovation and Development(No.2010Z0940)
文摘Objective： To explore the apoptosis mechanism of Wenxia Changfu Formula （温下肠腑方, WCF） in reversing drug resistance of lung cancer in vivo. Metbods： Thirty model mice were randomly assigned to three groups： control group, cisplatin （CDDP） group, and WCF group. A transplanted tumor model of lung adenocarcinoma was established in all groups. Mice in the WCF group received intragastric administration of WCF （0.2 mL/10 g body weight） everyday in addition to CDDP intraperitoneally （5 mg/kg body weight） twice a week. The mice in the CDDP group received CDDP intraperitoneally （5 mg/kg body weight） twice a week, while the control group received normal saline intraperitoneally （0.2 mL/10 g body weight） everyday. The weight of the nude mice and respective tumors, tumor volume and tumor-inhibiting rate were measured. Electron microscopy was used to observe the existence of apoptosis body. Apoptosis index （AI） was detected by TdT-mediated dUTP nick end labeling staining. The expression of Fas and FasL mRNA was investigated by reverse transcription polymerase chain reaction, while immunohistochemistry was applied to detect the protein expression of Fas and FasL, caspase-3 and caspase-activated DNase （CAD）, respectively. Results： Compared with CDDP group and control group, WCF could significantly reduce the tumor volume from the 19th day and alleviate the tumor weight （P〈0.05）, and the apoptosis body was found in tumor cells in the WCF group. WCF could also enhance the level of AI, up-regulate the expression of caspase apoptosis pathway related protein caspase-3 and CAD, as well as the expression of Fas, FasL mRNA and protein （P〈0.05）. Conclusion： WCF could improve the sensitivity of tumor cells to CDDP and reverse the drug resistance by inducinq the apoptosis.
文摘目的:探讨温下方对巨噬细胞极化调控机制及M2型巨噬细胞(M2-TAMs)对A549细胞侵袭转移的影响。方法:动物实验:BALB/c裸鼠随机分为模型组和温下方组,灌胃干预。活体成像分析光子强度;观察肺部病理变化;识别CD68/CD163双标M2-TAMs。细胞实验:佛波酯(PMA)及白介素-4(IL-4)诱导M2-TAMs;流式细胞术检测M2-TAMs标志蛋白CD163表达;PCR检测温下方对M2-TAMs相关基因mRNA表达的影响;划痕和Transwell检测温下方对M2-TAMs促A549细胞侵袭转移的影响;Western blot和PCR检测非受体型酪氨酸蛋白激酶3(J AK3)、信号转导及转录激活因子6(ST AT6)和过氧化物酶增殖物激活受体γ(PP A Rγ)蛋白及mRNA表达。结果:动物实验显示,与模型组比较,温下方在第28天~35天有效降低转移灶光子强度(P<0.05,P<0.01);温下方有效减轻肺部病理损伤;温下方降低转移瘤中M2-TAMs特异性表型CD68和CD163双阳表达。细胞实验显示,成功诱导的M2-TAMs高表达CD163;与M2组比较,温下方显著抑制M2-TAMs标志物Arg-1、Fizz-1、C D206 m R NA表达(P<0.01);与M2组比较,M2+WC F组显著降低M2-T A M s促A549细胞的迁移和侵袭能力(P<0.01);与M2组相比较,温下方有效降低p-JAK3、p-STAT6和PPARγ蛋白及JAK3、STAT6、PPARγmRNA表达(P<0.05,P<0.01)。结论:温下方可能通过调控JAK3/STAT6/PPARγ信号通路,抑制巨噬细胞M2极化,降低A549细胞侵袭转移。
基金Supported by the National Natural Science Foundation of China (No.81072832N0.30873222)Shandong Provincial Young and Middle-aged Scientists(No.2008BS03010)
文摘Objective： To observe the effect of the combination of Wenxia Changfu Formula （温下肠腑方, WCF） with cisplatin （CDDP） on inhibiting non-small cell lung cancer （NSCLC） in vitro and in vivo and explore its mechanism from its effect on cell cycle. Methods： In vitro, WCF-containing serum was prepared and the rhubarb bl, emodin, and aconitine were detected qualitatively by high-performance liquid chromatogram （HPLC）. A549 cell lines were treated with blank control （dimethyl sulfoxide）, normal serum, normal serum with CDDP （1.25, 2.5, and 5.0 μg/mL, respectively）, WCF-containing serum plus different doses of CDDP （1.25, 2.5, and 5.0 μ g/mL, respectively）. The inhibitory effect was detected by 3-（4,5）-dimethylthiazo（-z- yl）-3,5-diphenylterazolium bromide （MI-I）. The cell cycle was detected by flow cytometry. The protein and mRNA expressions of cyciin D1, proliferating cell nuclear antigen （PCNA）, retinoblastoma （Rb）, and p16 were observed with immunofluorescence and RT-PCR, respectively. In vivo, nude mice xenograft model was established and grouped into the control, CDDP, WCF, and combination groups. The combination＇s inhibition of tumor growth and influence on the weight, spleen, and thymus gland were observed. Results： The inhibitory rate of the combination against A549 cell lines excelled the CDDP alone significantly （P〈0.05）; the combination showed a synergism inhibitory effect （Q=1.19）. Compared with the monotherapy, the combination increased the cell percentage in G0/G1 phase and decreased the cell percentage in S phase significantly （P〈0.05）; the protein and mRNA expressions of cyclin D1, PCNA, and Rb were significantly reduced; the protein and mRNA expressions of p16 were significantly enhanced. Compared with the monotherapy, the combination inhibited the tumor growth significantly in vivo and reduced the weight of tumor （P〈0.05）; compared with the CDDP group, the spleen and thymus gland index of the combination group were enhanced significantly （P〈0.05）. Conclusions： The combination of WCF with CDDP significantly inhibited the A549 cell lines proliferation in vitro and the growth of the tumor in vivo; it inhibited effectively the atrophy of the immune organ caused by chemotherapy. The combination inhibited overproliferation of A549 cell lines by arresting the Go/G1 phase of cell cycle and affecting the protein and mRNA expressions of cell cycle-related proteins, cyclin D1, etc.
