清胰化积方对胰腺癌CFPAC1移植瘤裸小鼠血清白细胞介素6、白细胞介素8及肿瘤坏死因子α表达的影响

　　　　 作者：欧阳华强， 刘鲁明， 陈震， 罗建民， 于尔辛

【摘要】 　　目的：观察清胰化积(Qingyi Huaji， QYHJ)方对人胰腺癌CFPAC1细胞系裸小鼠移植瘤的抑瘤作用及血清白细胞介素6（interleukin6，IL6）、白细胞介素8（interleukin8，IL8）和肿瘤坏死因子α（tumor necrosis factorα，TNFα）表达水平的影响。方法：40只裸小鼠右前肢腋部皮下移植胰腺癌瘤块建立荷瘤模型。造模成功的裸小鼠随机分为模型组、卡培他滨组、低剂量(36 g/kg)清胰化积方组和高剂量(72 g/kg)清胰化积方组，每组10只，分别采用含阿拉伯胶的柠檬酸缓冲液、卡培他滨混悬液及清胰化积方药液灌胃。治疗5周后，眼眶取血，酶联免疫吸附测定法检测血清IL6、IL8和TNFα含量；脱颈椎处死裸小鼠，剥取肿瘤，称取质量，测量肿瘤直径，计算抑瘤率。结果：低剂量清胰化积方组CFPAC1裸小鼠移植瘤质量下降，与模型组比较，差异有统计学意义(P&<0.05)。高、低剂量清胰化积方组裸小鼠血清IL6及TNFα水平显著低于模型组（P&<0.01），且低剂量清胰化积方组和卡培他滨组血清IL8水平低于模型组(P&<0.05)。相关分析显示，裸小鼠移植瘤质量与血清IL6、IL8和TNFα水平呈线性正相关(P&<0.01)。结论：常规剂量的清胰化积方对裸小鼠胰腺癌移植瘤模型有明显的抑瘤作用，其作用机制可能与下调细胞因子IL6、IL8和TNFα的表达有关。

【关键词】 胰腺癌; 复方; 白细胞介素6; 白细胞介素8; 肿瘤坏死因子α; 裸小鼠

　　Pancreatic carcinoma is a common malignant tumor in digestive system with rapid progress and high incidence of metastasis. At the time of diagnosis， most patients with pacreatic carcinoma have already been at the advanced stage with a 5year survival rate less than 5%［1］. A study in Cancer Hospital of Fudan University with 134 patients of advanced pancreatic cancer suggested an effective tumor inhibition role of Qingyi Huaji (QYHJ) decoction， a compound traditional herbal medicine. The results showed that the 1， 3 and 5year survival rates were 25.0%， 14.1% and 8.4%， respectively， with a median survival time of 7.6 months. Sixteen patients survived for more than 3 years (5 survived for more than 5 years) ［2， 3］. The survival rates were higher than the results in other similar studies with Western medicine. Although indicated with an effective tumor inhibition function， the mechanism of QYHJ decoction has hot been further explored yet. It has been reported that serum cytokine levels and its immunomodulatory effects are closely related to the development of pancreatic cancer［4］. In this study， we investigated the effects of QYHJ decoction on tumor inhibition and serum levels of interleukin6 (IL6)， interleukin8 (IL8) and tumor necrosis factorα (TNFα) in nude mice with transplanted tumors of human pancreatic cancer.

　　1 Materials and methods

　　1.1 Experimental materials

　　1.1.1 Cell culture and animals

　　Human pancreatic adenocarcinoma cell line CFPAC1 was purchased from the Institute of Biochemistry and Cell Biology， Shanghai Institute for Biological Sciences， Chinese Academy of Sciences. According to the protocol of American Type Culture Collection (ATCC)［5］， the cell line was cultured in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal bovine serum， sodium pyruvate and 4(2hydroxyethyl)1piperazineethanesulfonic acid (HEPES) in a humidified atmosphere containing 5% CO2 at 37 ℃. Female 5weekold BALB/cnu/nu mice， SPF grade (Certification No. 122)， weighing from 18 to 22 g， were purchased from Shanghai Experimental Animal Center of the Chinese Academy of Sciences.

　　1.1.2 Therapeutic medication

　　QYHJ decoction， composed of Baihuasheshecao (Herba Hedyotidis) 30 g， Banzhilian (Herba Scutellariae Barbatae) 30 g， Moyu (Rhizoma Amorphophalli Rivieri) 30 g， Jiaogulan (Herba seu Radix Gynostemmatis) 30 g and Baidoukou (Fructus Amomi Rotundus) 6 g， was provided by Jiangyin Tianjiang Pharmaceutical Co.， Ltd. (Jiangsu， China). According to body surface area conversion table between humans and mice， herb medication were prepared in QYHJ decoction groups at a dose of 36 g/kg or 72 g/kg， which including the crude drug of 1.8 g/mL and 3.6 g/mL， respectively， and oral administration was 0.4 mL/d per 20 g body weight for nude mice. Capecitabine (F. HoVmanLa Roche， Shanghai， 0.5 g/tablet， certification No. h30073024) was crushed and dissolved in 100 mL citrate buffer (pH 6.0， 40 mmol/L) containing 5% (w/v) Arabic gum［6］.

　　1.1.3 Main reagents and instruments

　　Sunrise F039300A microplate reader was provided by TECAN， Switzerland; enzymelinked immunosorbent assay (ELISA) kits of IL6， IL8 and TNFα were purchased from Shanghai Senxiong BioTechnology Co.， Ltd.， China; IMDM was purchased from Gibco Company， USA， (lot No. 12200036); fetal bovine serum was provided by PAA Company， Austria (lot No. A151081479).

　　1.2 Methods

　　1.2.1 Preparation of mice model

　　CFPAC1 cells in logarithmic growth phase were prepared as single cell suspension at a concentration of 1×107 cells/mL and were subcutaneously inoculated into the right flank of five nude mice at a dose of 2×106 cells. Three weeks later， mice were sacrificed by cervical dislocation when the xenografts size reached 1 cm in diameter. Tumors were excised from mice， cut into small pieces and transplanted subcutaneously into the right forelimb armpit of another 40 nude mice.

　　1.2.2 Experimental grouping and medication

　　Seven days after transplantation， as the mean size of the tumors was about 0.4 cm in diameter， mice were randomly pided into untreated group (Arabic gum)， capecitabine group， lowdose QYHJ decoction group (36 g/kg) and highdose QYHJ decoction group (72 g/kg)， with 10 mice in each group. For untreated group， citrate buffer solution (containing 5% Arabic gum) was administered by gavage， 0.4 mL per mouse， once a day for 5 weeks; for capecitabine group， capecitabine suspension 0.4 mL per mouse by gavage， once a day， 5 days per week， 3 weeks continuously［7］; for lowdose QYHJ decoction group， 1.8 g/mL QYHJ 0.4 mL per mouse (36 g/kg)， once a day for 5 weeks; for highdose QYHJ decoction group， 3.6 g/mL QYHJ 0.4 mL per mouse (72 g/kg)， once a day for 5 weeks［8］.

　　1.3 Experimental detection

　　1.3.1 Observation of general condition

　　Fur color， motility， nutritional status， body weight， mental condition and tumor size of the nude mice were observed and recorded daily.

　　1.3.2 Tumor volume changes

　　The maximal and minimal diameters of the tumor were measured by a vernier caliper once a week. Tumor volume and the inhibition rate were calculated according to calculation formula of tumor volume (V): V=1/2 (maximal diameter×minimal diameter2) and inhibition rate of tumor volume=(1－average tumor volume of treatment group/average tumor volume of control group)×100%. The growth curve was drawn according to the tumor volume measured daily.

　　1.3.3 Tumor inhibition rate

　　Mice were sacrificed 1 day after the last time of treatment. Tumor weight was estimated and the tumor inhibition rate was calculated. Tumor growth inhibition rate=(1－average tumor weight of treatment group/average tumor weight of control group)×100%。

　　1.3.4 Detection of serum levels of IL6， IL8 and TNFα Concentrations of serum IL6， IL8 and TNFα were examined by ELISA， using blood sample from eye socket which was stored at room temperature for 30 min， centrifuged (1 000×g) for 15 min， and then cryopreserved at －80 ℃.

　　1.4 Statistical analysis

　　Statistical analysis was carried out with STATA software (version 10.0， College Station， Texas， USA). The measurement data were presented as x±s. One way analysis of variance was used to compare the differences between groups (Symmetry transformation was used for nonnormal distribution or variance nonhomogeneity of the data)， and the pairwise comparison was performed with Sidak method. Linear regression analysis was used to evaluate the correlation between cytokine level and tumor weights. Scatterplot graphs were created， and regression equations were built up. For all tests， P values less than 0.05 were considered statistically significant.

　　2 Results

　　2.1 General condition of the nude mice

　　During the first four weeks after successful modeling， nude mice in each group had balanced appearances， agile movements， flexible responses and increased body weights. As the tumor volume increased， life status of the mice worsened gradually， especially for untreated group. Two mice in the untreated group appeared as dark fur， loss of weight and energy at the fifth week. No deaths were observed until the mice were sacrificed. The whole body weight changes of nude mice showed an increasing tendency， however without significant differences between groups at each time point (P&>0.05， Table 1).Table 1 Body weight of nude mice in four groups(略)

　　2.2 Effects of QYHJ decoction on volume of transplanted tumors

　　Tumor with size of grain rice， could be detected immediately after the tumor pieces were subcutaneously transplanted into the nude mice. Primary tumor mass size was measurable after one week， with 100% successful modeling. At the initial stage， transplanted tumors were characterized with slow growth， spherical or hemispherical appearance， and movable. The tumors became harder gradually， with different inpidual size. After treatment， the average volumes of tumor in the capecitabine group， and lowdose and highdose QYHJ groups were less than that in the untreated group， and the inhibition rates of tumor volume were 38.24%， 16.18% and 13.24% in those three groups respectively. There was no statistical difference for tumor volume between groups (P&>0.05， Table 2 and Figure 1). Table 2 Tumor volumes and growthinhibition rates of nude mice in different groups（略）

　　2.3 Effects of QYHJ decoction on tumor weights

　　Tumor weight decreased significantly in the capecitabine and lowdose QYHJ groups as compared with the untreated group (P&<0.05)， while there were no significant differences between the highdose QYHJ group and the untreated group (P&>0.05) and between the QYHJ groups and the capecitabine group (P&>0.05， Table 3).Table 3 Tumor weights and growthinhibition rates of nude mice in different groups（略）

　　2.4 Effects of QYHJ decoction on serum levels of IL6， IL8 and TNFα Compared with the untreated group， serum levels of IL6 and TNFα in the high and lowdose QYHJ groups decreased significantly (P&<0.01)， and so did the serum IL8 levels in the capecitabine and lowdose QYHJ groups (P&<0.05). Compared with the capecitabine group， IL6 and TNFα levels in the high and lowdose QYHJ groups also decreased significantly (P&<0.01)， however， IL8 level in the lowdose QYHJ group increased without significance (P&>0.05， Table 4).Table 4 Contents of serum IL6， IL8 and TNFα of nude mice in different groups(略)

　　2.5 Correlation of tumor weights and serum levels of IL6， IL8 and TNFα As mentioned above， there were statistical differences in serum levels of IL6， IL8 and TNFα between the QYHJ groups and the untreated group. The relationships between transplanted tumor weight and serum levels of cytokines were further analyzed. The result showed a positive linear correlation between tumor weight and serum level of IL6 in all groups (R2=0.32， P&<0.01). The regression equation for serum level of IL6 and tumor weight was =12.8+23.58x (Figure 2， x was tumor weight; was estimate of corresponding cytokine level， similarly hereinafter). There was also a positive linear correlation between serum level of IL8 and tumor weight (R2=0.76， P&<0.01)， and the regression equation was =91.51+201.08x (Figure 3). The regression equation for serum level of TNFα and tumor weight was =15.93+17.91x (R2=0.43， P&<0.01， Figure 4).

　　3 Discussion

　　QYHJ decoction is a Chinese herbal compound based on the pathological mechanism of “noxious dampness， toxic heat and accumulation of pathogenic dampness， heat and toxin” for pancreatic cancer in traditional Chinese medicine［9］. The product is made from Chinese herbs by using Moyu as monarch drug， which can reduce phlegm and resolve masses， detoxify and relieve swellings， dissipate blood stasis and relieve dyspepsia. Both Baihuasheshecao and Banzhilian are used as ministerial drugs， with the function of heatclearing and detoxifying， removing dampness and deswelling， and activating blood analgesia. Jiaogulan is used as an adjuvant drug of strengthening healthy qi and disintoxication， resolving phlegm and anticancer. Baidoukou is used as the messenger drug for dissipating dampness to normalize stomach and promoting qi circulation to alleviate middle energizer. Clinical studies showed that QYHJ decoction was effective in advanced pancreatic cancer patients by heatclearing and detoxifying， removing dampness and deswelling， regulating qi and dissipating blood stasis［2， 3］. We previously found that QYHJ decoction could inhibit the proliferation of human pancreatic cancer cell line SW1990 in vivo， and tumor weight in QYHJ group was significantly lower than that in the negative control group (P&<0.05)［8］. When combined with gemcitabine， the tumor inhibition rate reached 92.69% to 93.41%， and tumor weight was significantly lower in the combined treatment group when compared with the gemcitabine group (P&<0.05)［10］.

　　The results suggested that inhibition rates of tumor weight in the low and highdose QYHJ groups were 34.4% and 32.8% respectively， with statistical significance for the lowdose group when compared with the untreated group (P&<0.05). However， inhibition rates of tumor volume were 16.2% and 13.2% respectively， without statistical significance between each QYHJ group and the untreated group (P&>0.05). Moreover， heterogeneous changes were noticed in the capecitabine group and the QYHJ group with central necrosis， but no obvious tumor necrosis was detected in the untreated group. Therefore， disproportionate relation of tumor volume and weight may be due to internal necrosis in some tumor tissues or the possibility of measurement errors in the body surface.

　　The results showed that serum levels of IL6， IL8 and TNFα in the QYHJ groups were significantly lower than those in the untreated group (P&<0.01， P&<0.05). TNFα is also called cachectin. It is a monokine mainly produced by mononuclear macrophages， which can not only selectively kill some tumor cells and induce their apoptosis， but also has an effect of multiple immunoregulation. TNFα receptors are widely distributed throughout tumor cells and blood cells. Binding of TNFα and its receptors from some tumor cell surface of membrane may lead to death of these cells［11］. High serum TNFα level is an adverse prognostic factor for survival and eventfree survival in patients with untreated acute myeloid leukemia or highrisk myelodysplastic syndromes［12］. TalarWojnarowska et al［13］ studied 41 patients with pancreatic adenocarcinoma， 56 with chronic pancreatitis and 50 healthy volunteers. Peripheral venous blood samples were obtained from all patients for TNFα serum concentration measurement. The results showed that plasma level of TNFα was significantly higher in pancreatic cancer patients (32.7 pg/mL) than in chronic pancreatitis patients (3.2 pg/mL) and healthy people (&<1.6 pg/mL， P&<0.01). A positive correlation between weight loss in pancreatic cancer patients and serum level of TNFα (P=0.02) was also observed in their study. Patients with higher serum TNFα level were more likely to develop cachexia. In the present study， serum TNFα content in the untreated group was the highest， followed by capecitabine group. The results suggested that mechanism of capecitabine in the treatment of pancreatic cancer might have no relation to TNFα level， which was similar to the conclusion made by Saif et al［14］ in a phase Ⅰ clinical trial of capecitabine and concurrent radiotherapy for locally advanced pancreatic cancer.

　　TNFα has the function of killing or inhibiting cancer cells， but in current study we observed that the higher the serum TNFα level， the heavier the tumor weight， which indicated an acceleration of TNFα secretion accompanying with the growth of tumor. As the tumor volume increased， serum TNFα level showed an increasing trend accordingly; and the decreased TNFα level revealed that tumor was under control to a certain extent. Serum TNFα level and its relationship with tumor progression remains to be elucidated.

　　IL6 is a cytokine with the function of multiple immunoregulation. Many tumor cells may produce and secrete IL6. Some studies showed that 174G/C IL6 gene polymorphism influenced circulating IL6 levels. Increased IL6 serum level may be positively correlated with tumor size and the presence of liver metastases in patients with pancreatic adenocarcinoma (P&<0.01)［15］， and negatively correlated with serum albumin level. Overexpression of IL6 usually means poor status and prognosis of cancer patients［4］. IL8 is also called CXC chemokine ligand8， which is a cytokine mainly secreted by macrophages and epithelial cells， etc［16］. IL8 may promote tumor growth directly or through its role of stimulating angiogenesis. Significance of serum IL8 in pancreatic cancer is similar to IL6. Overexpression of IL8 is closely related to weight loss， and these patients are prone to cachexia rapidly. However， some researches showed that the correlation of IL8 and survival of patients was not so notable when compared with IL6［4］.

　　In the present study， serum IL6 level in untreated group was the highest. No significant difference was found between the capecitabine group and the untreated group， while the difference was statistically significant in the low and highdose QYHJ groups as compared with the untreated group (P&<0.01)， especially for the lowdose QYHJ group. The results indicated that conventional dose of QYHJ decoction could downregulate the expression of serum IL6 in CFPAC1 transplanted tumorbearing mice， but the downward effect was not directly proportional to dosage of QYHJ decoction. Moreover， we have observed that serum level of IL8 was linearly positively correlated with transplanted tumor weight of the mice (R2=0.76， P&<0.01)， and relationship between tumor weight and IL8 level was more close than its relation with IL6 or TNFα.

　　In the process of tumor development， high expression levels of cytokines such as IL6， IL8 and TNFα are usually signals of progression of disease or poor prognosis. They are closely related to the occurrence of cancer cachexia. In the present study， serum IL6， IL8 and TNFα levels in transplanted tumor of human pancreatic cancer cell line CFPAC1 in nude mice were linearly positively correlated with tumor weight. As tumor weights in QYHJ groups were significantly lower than that in the untreated group， cytokine levels were correspondingly lower. The results demonstrated that serum levels of IL6， IL8 and TNFα were important indicators for evaluating the sensitivity of pancreatic cancer to treatment of QYHJ decoction.

　　In conclusion， conventional dose of QYHJ decoction (36 g/kg) has an obvious antitumor effect on the subcutaneously transplanted tumors of human pancreatic carcinoma in nude mice. The mechanism may be related to downregulation of cytokines such as IL6， IL8 and TNFα.

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