Effect of Qinggan Huayu granule on H22 liver ascitic tumor mice

Fei-Ya Suo, Xiao-Ran Zhu, Zhen-Huan Yang, Shu-Kun Yao

1. Graduate School, Beijing University of Chinese Medicine, Beijing 100029, China

2. Department of Gastroenterology, China-Japanese Friendship Hospital, Beijing 100029, China

Keywords:H22 liver cancer ascites tumor Mouse Qinggan Huayu granule Survival period Peritoneal vascular permeability

ABSTRACT Objective: To evaluate the therapeutic effect of Qinggan Huayu granule on mice with H22 liver cancer ascites tumor. Methods: A H22 liver cancer ascites mouse model was established by intraperitoneally injecting H22 liver cancer cells. Mice were randomly divided into the model group, the Ganfule group (1.35 g/kg), the fluorouracil group (50 mg/kg i.p), the Qinggan Huayu granule groups at low(0.67 g/kg), medium (1.34 g/kg), and high (2.68 g/kg) doses. Then the mice were administered continuously for 10 days and body weight and abdominal circumference were monitored every 3 days. On day 11, eight rats in each group were randomly selected for dissection to detect the amount of peritoneal water, peritoneal permeability and histopathological changes. The remaining mice were observed for survival.In addition, the vascular endothelial growth factor A (VEGFA) and vascular endothelial growth factor receptor 2 (VEGFR2) were determined by Western blotting. Results: Compared with the model group, the weight growth of mice in the fluorouracil group and the medium-dose and high-dose Qinggan Huayu granule groups was slower (P < 0.05). Moreover, the abdominal circumference of mice in each treatment group was increased slowly. There were significant differences in abdominal circumference between the fluorouracil group, the medium-dose group and the control group from day 6 (P < 0.05) while the abdominal circumference of the high dose group was significantly smaller than that of the control group from day 12 (P< 0.05). Moreover, compared with the model group, the amount of ascites in the mediumand high-dose Qinggan Huayu granule groups was decreased significantly (P < 0.05). The optical density value of ascites supernatant in medium- and high-dose Qinggan Huayu granule group and the fluorouracil group decreased significantly (P < 0.05) and the survival period of the medium-dose Qinggan Huayu granule group and the fluorouracil group was prolonged prominently (P < 0.05). There was no significant difference in the low-dose Qinggan Huayu granule group and the Ganfule group. Peritoneal histopathological assay showed more complete peritoneal structure, less edema and less angiogenesis of the peritoneum in the fluorouracil group and the medium- and high-dose Qinggan Huayu granule group, which was better than that of the Ganfule group and the low-dose group. Compared with the model group, the expressions of VEGFA and VEGFR2 in the medium-dose Qinggan Huayu granule group decreased significantly (P < 0.05, P < 0.01). Conclusion: Qinggan Huayu granule can inhibit ascites production in the mice model with H22 liver cancer ascites tumor, prolong the survival of mice, and reduce peritoneal permeability and suppress the increase of peritoneal neovascularization. The mechanism may be related to the inhibition of VEGF /VEGFR pathway.

1. Introduction

Primary liver cancer is the fourth most common malignant tumor in China and the second most deadly cause of cancer. It seriously threatens the life and health of Chinese people [1].Ascites of liver cancer is one of the most common complications of advanced liver cancer, which can cause abdominal distension, abdominal pain, fatigue, poor appetite, dyspnea, restricted activity, circulation disorders and multiple organ failure, etc., seriously affecting patients' quality of life, difficult clinical treatment, poor prognosis,and no clear clinical treatment plan[2-4].Chinese medicine treatment can improve the symptoms mentioned above, improve the body resistance, improve the quality of life, effectively control the further growth of ascites, prolong the survival of patients [1, 5].Therefore,it is of great significance to study the treatment of malignant ascites with Chinese medicine[6-7]. Qinggan Huayu recipe is carried out by professor Yao Shukun preliminary research results confirmed that the Qinggan Huayu recipe circulation can significantly improve the immune function of patients with primary liver cancer, high quality of life and prolong the survival period, with very high value of the development of research and development, and thus on the basis of the development the "Qinggan Huayu granule" for new drug research and development and clinical promotion[8-10].This study evaluated the efficacy and discussed the basic mechanism of Qinggan Huayu granule in the treatment of H22 hepatocellular carcinoma ascites tumor mice, providing pharmacodynamic basis for the development of new drugs, and providing basic experimental support for the treatment of primary liver cancer patients with malignant ascites.

2. Materials and reagents

2.1 Main experimental materials

Mouse hepatoma cell H22, purchased from Institute of Basic Medicine, Chinese Academy of Medical Sciences;Male ICR mice, aged 4 weeks and weighing about 20g, were provided by Beijing Huafukang Biotechnology Co., LTD., license no. SCXK(Beijing) 2019-0008, kept in SPF Animal Laboratory of China-Japan Friendship Hospital.The experiment conforms to the ethical regulations of animal experiment, ethical code:190114.

2.2 Medicinal materials and reagents

Fluorouracil injection, purchased from Tianjin Jinyao Pharmaceutical Co., LTD., Batch Number 1504261;0.9%normal saline injection, purchased from Beijing Double Crane Pharmaceutical Co., LTD., Batch No. 1003182; Ganfule capsule,purchased from Hunan Jiudian Pharmaceutical Co., LTD Company,Batch Number Z20050817; Qinggan Huayu Granule dry powder(batch no.:20170731) is provided by department of Pharmacy,China-Japan Friendship Hospital. "Dry powder" refers to the extract of the test substance, and each gram of the extract is equivalent to 4.472g crude drug.

2.3 Establishment of mouse H22 liver cancer ascites tumor model

2.3.1 Preparation of H22 liver cancer cellsThe cells were quickly resuscitated by water bath at 37℃, PBS was added and centrifuged to remove the frozen liquid, and physiological saline was added for resuscitation. The cells were prepared with a concentration of 1×107cells/ml.

2.3.2 Intraperitoneal passage of mouse H22 hepatoma cells

Three SPF male ICR mice aged 4 weeks were injected with 0.2ml prepared cell suspension in the right lower abdominal cavity and fed in SPF animal chamber. The growth and abdominal changes of the mice were observed daily. One week later, ascites were extracted and cell count was performed in super clean workbench, and the cell concentration was adjusted to 2×107/ml.The intraperitoneal cells of mice were subcultured by the same method for 2 times.After the third passage, an appropriate amount of ascites was taken and counted. The survival rate of cells was above 98%, and the cell concentration was adjusted to 2×107/ml.

2.4 Establishment of mouse H22 liver cancer ascites tumor model

A total of 120 SPF grade male ICR mice were selected at the age of 4 weeks, weighing about 20g.The mice were injected into the right lower abdominal cavity aseptically 0.2ml of prepared cell suspension was used to establish the mouse model of H22 hepatoma ascites tumor. After inoculation, the mice were observed and fed in the SPF animal laboratory.

2.5 Grouping and administration

Twenty-four hours after inoculation, 120 mice were randomly divided into model group, Qinggan Huayu granule low-dose,medium-dose and high-dose groups, Ganfule group and fluorouracil group, with 20 mice in each group. Model group was given aseptic water once a day, Qinggan Huayu granule low-dose group (0.67g/kg), Qinggan Huayu granule medium-dose group (1.34g/kg),Qinggan Huayu granule high-dose group (2.68g/kg), ganforle group(1.35g/kg), the above groups were given intragastric administration once a day, and fluorouracil group (50mg/kg)Intraperitoneal injection was given once every 7 days, and the drugs were stopped for 10 days in the above groups.

2.6 Observation Indicators

2.6.1 General condition and body weight and abdominal circumference of mice were monitored

The general conditions of mice in each group were monitored,including appearance, activity and mental state, and their body weight and abdominal circumference were measured every 3 days.

2.6.2 Abdominal water volume, ascites inhibition rate and peritoneal permeability of mice

Twenty-four hours after the end of medication, 8 mice in each group were randomly selected and injected 0.2 mL 5% Evanblue solution into the tail vein. After 2 hours, the mice were sacrificed to extract ascites, and the volume of ascites was measured, and the inhibition rate of ascites was calculated. The supernatant of ascites was centrifuged, and the absorbance (OD) (wavelength 540nm)was measured by ELISA. Then the peritoneal permeability was calculated. Ascites inhibition rate = (1- average abdominal water volume in the treatment group/average abdominal water volume in the model group) ×100%. Ascites permeability: The peritoneal vessel permeability was measured by the absorbance OD value of Evanblue.

2.6.3 Peritoneal tissue pathological staining of miceThe above mice with ascites extraction were taken, the abdominal epidermis was peeled off, and a 1cm×1cm piece of peritoneum was cut off from the left middle and lower abdomen. After washing with normal saline, the samples were placed in an embedding box to prevent sample wrinkling, and the samples were fixed in formalin solution together with the embedding box. Subsequent pathological H-E staining section observation was conducted.

2.6.4 Survival observationThe natural survival days of the remaining 12 mice in each group were recorded, the average survival days and median survival time were calculated, the survival curve was drawn, and the life extension rate was calculated. Life extension rate = (average survival days in treatment group -average survival days in control group)/average survival days in model group x 100%.

2.7 Western Bolt detection of peritoneal tissue

According to the pharmacodynamics evaluation, the optimal dosage group of Qinggan Huayu granule was determined. The expression quantity of VEGFA and VEGFR2 in peritoneum of mice in this group and model group were detected by Western Blot. The peritoneal tissues were cleaved, protein quantified, denaturated,electroporated, transformed, and sealed. VEGFA antibodies(AB1316, ABCAM) and VEGFR2 antibodies (9698S, CST) were incubated overnight at 4℃. The corresponding secondary antibodies were incubated and imaging was performed. The gray value of the strip was analyzed by ImageJ V1.53C software, and the gray value was compared with the internal reference strip to obtain the relative expression.

2.8 Statistical Methods

SPSS 20.0 statistical software was used for statistical analysis of all data obtained, and the measurement data were expressed as mean ± standard deviation (x± s). If the variance was uniform, oneway ANOVA was used for overall comparison, and Dunnet's test was used for pairwise comparison. If variance is not uniform, nonparametric test is used for overall comparison, and Kruskal-Wallis test is used for pair comparison.

3. Results

3.1 General condition observation of mice

After inoculation with H22 cells, all mice showed dark and yellow hair, clumped and knotted hair, reduced activity speed, deep concave eyes, abdominal distension and other manifestations. Compared with model group, the above symptoms appeared later in all drug groups,especially in fluorouracil group, Qinggan Huayu granule mediumdose and high-dose groups.

3.2 Body weight monitoring of mice

Compared with the model group, the weight growth of mice in each treatment group was slow, among which the weight growth trend of fluorouracil group, Qinggan Huayu medium and high dose group was significantly slow (P < 0.01, P < 0.05). There was no statistical significance in the weight of mice in Qinggan Huayu low dose group and Ganfule group compared with the model group (P > 0.05),which was showed in Table 1.

3.3. Abdominal circumference monitoring of mice

Compared with model group, abdominal circumference of mice in all drug treatment groups increased slowly, among which fluorouracil group and medium-dose group showed significant difference from the 6th day (P < 0.01), and high-dose group showed difference from the 12th day (P < 0.05).There was no difference in body weight between qinggan huayu low-dose group and Ganfule group (P > 0.05). Abdominal circumference was showed in table 2.

Table1 Body weight of the mice in each group (g,x±s,n=12)

3.4 Abdominal water volume, ascites inhibition rate and peritoneal permeability of mice

Compared with model group, the abdominal water volume in Qinggan Huayu medium-dose group and high-dose group was less(P < 0.05), and the ascites inhibition rate was 25.67% and 19.28%respectively. There was no statistical difference in abdominal water volume in qinggan Huayu low-dose group and Ganfule group.Compared with the model group, the OD value of qinggan Huayu medium-dose, high-dose and fluorouracil groups significantly decreased (P < 0.05), suggesting that the medium-dose and highdose treatment of Qinggan Huayu granules can inhibit the growth of ascites in H22 ascites tumor mice and reduce peritoneal permeability.The above were showed in Table 3.

3.5 H-E staining results of peritoneal histopathology in mice

The peritoneal tissues of 8 mice in each group were pathologically stained and observed. It was found that peritoneal cells in the model group were arranged loosely and changed in a broken flock-like manner, with neovascularization, accompanied by a large number of neutrophil infiltration, and a large number of cell degeneration and necrosis. In the low dose group of qinggan huayu, peritoneal tissue cells were arranged loosely, neovascularization appeared,accompanied by a large number of neutrophil infiltration, and some cells were necrotic. In qinggan huayu medium dose group, the tissue cell structure was stable, mild edema, and the number of neutrophils was small. The high-dose group had stable tissue cell structure, mild edema and more neutrophils. The cell structure of Ganfule group was loose, edema was obvious, the number of neutrophils increased significantly, and peritoneal thickness increased significantly. In the fluorouracil group, the cell morphology and structure were normal and neatly arranged, with only slight edema. The above results suggest that the peritoneal structure of mice in the fluorouracil group is the most complete, the edema is slight, and no new blood vessels are observed. It is considered that chemotherapy drugs directly contact cancer ascites, and the effect is the best. Compared with model group, the peritoneal structure of mice in Qinggan huayu medium-dose and high-dose groups was more complete, edema was lighter, and angiogenesis was less, which was better than that in Ganfule group and Qinggan Huayu low-dose group. The above was shown in figure 1.

Figure 1 H-E staining results of peritoneal histopathology of mice in each group

3.6 Survival of mice

Compared with model group, the average survival period of Qinggan Huayu granule medium-dose group were significantly longer (P < 0.05), and the average survival period of fluorouracil group were significantly longer (P < 0.01).The life extension rate of qinggan Huayu granule low-dose group was16.7%, 25.7% in qinggan Huayu granule medium-dose group, 15.3% in Qinggan Huayu granule high-dose group, 18.1% in Ganfule group, and66.7% in fluorouracil group, among which the medium-dose group and fluorouracil group could effectively prolong the survival period.The difference was statistically significant. The median survival time of model group and qinggan huayu low-dose, medium-dose and high-dose groups was 14,16,18,16 days respectively. Compared with model group, the survival period of qinggan huayu group was prolonged, and there was statistical difference in qinggan Huayu middle dose group (P < 0.05). The above was showed in Table 4 and Figure 2.

Table 2 Abdominal circumference of mice in each group (cm,x±s,n=12)

Table 3 Amount and inhibition rate of ascites and OD value of ascites supernatant of mice in each group (x±s,n=8)

Figure 2 Survival curve of the mice

3.7 Western Blot detection

Western Blot was performed on peritoneal tissues of mice in model group and qinggan huayu medium-dose group. The detection results are shown in Figure 3. The expression levels of VEGFA and VEGFR2 in peritoneum of mice in qinggan huayu group were significantly lower than those in model group, with statistical significance (P < 0.05, P < 0.01).

Figure 3 Expression of VEGFA and VEGFR2 in the model group and the middle-dose Qinggan Huayu group

Table 4 Survival of mice in each group (x±s,n=12)

4. Conclusion

The medium and high dose of Qinggan Huayu granules can improve the general condition of H22 hepatocellular carcinoma ascites tumor mice, slow down the weight growth of mice, reduce the formation of ascites, reduce peritoneal permeability, reduce peritoneal lesions and inhibit neovascularization. Among them,medium dose can prolong survival. The mechanism may be related to the inhibition of VEGF/VEGFR pathway.

In summary, ascites is one of the common complications of advanced primary liver cancer. Ascites of primary liver cancer is recurrent and refractory, and has always been a difficult problem in clinical treatment of liver cancer[11].At present, the treatment of liver cancer ascites lacks the support of evidence-based medical evidence,mainly including a variety of palliative measures, but the effect is not ideal, and there are many contraindications and too many side effects [12][13].The formation mechanism of ascites in liver cancer is complex and not completely clear. In addition to the decrease of albumin caused by abnormal liver function and leakage of tissue fluid caused by portal hypertension, the increase of peritoneal vascular permeability and peritoneal neovascularization caused by tumor invasion are considered to be one of the main causes of ascites formation. Therefore, reducing peritoneal microvascular permeability and inhibiting peritoneal angiogenesis are also considered as important strategies to reduce malignant ascites[14].Researchers have been trying to apply targeted drugs such as Bevacizumab and human recombinant vascular endothelial system suppression, which showed that they can get better in the near future curative effect, but the long-term curative effect is not exact, and drug prices was too high,survival in patients with difficult to significantly extend, effective treatment still need further study [15-18].

Ascites of primary liver cancer belongs to the category of"distention" in Traditional Chinese medicine. Professor Yao Shukun thinks that patients with liver cancer ascites can see pain on both flanks and foul breath. The extracted ascites is yellow and turbid,the temperature of abdominal epidermis increases, the tongue is red and greasy, and the pulse string is slippery. The local syndrome differentiation belongs to fever. The overall syndrome differentiation is that damp heat and blood stasis are blazing, accompanied by deficiency of Qi and blood. The main pathogenesis is that the cancer virus invades the tunnel. The tunnel is blocked and stagnates to generate damp heat. The stagnation of damp heat, toxin and blood stasis for a long time will block the operation of Qi and blood and the transmission of body fluid, Make the triple energizer resolve the imbalance, stop the water and liquid from gathering in the abdomen,and make the abdomen swell. In the late stage of the disease, the patient is full of positive deficiency and evil, and both qi and yang are damaged. However, the patient usually drinks alcohol or eats high calorie food supplements, which aggravates the stagnation of dampness, heat, toxin and blood stasis. "Su Wen" said: "when the damp and heat beat, the ruffian diaphragm will be depressed,and the urination will be unfavorable and edema will also occur","if the blood stasis does not go, the water will become", which explained that the damp and heat, blood stasis and Qi blocking mechanism are the key pathogenesis of water Qi disease. On this basis, Professor Yao Shukun put forward the treatment method of"Dispelling damp and heat and blood stasis to benefit liver cancer ascites", that is, to get rid of the damp and heat toxin, blood stasis and excess evil of Bi blocking, dredge the Sanjiao waterway, assist the movement of Qi and blood, and improve the distribution of body fluid, It can eliminate dampness and blood stasis without damaging Yang, regulate qi and water without damaging Yin. Therefore, it is concluded that Qinggan Huayu formula is suitable for the treatment of patients with middle and late stage and end stage of primary liver cancer, who lose the opportunity of chemotherapy, or complicated with a variety of complications, such as abnormal liver function and malignant ascites. It has achieved good results in early clinical practice. Qinggan Huayu granule takes Scutellaria baicalensis and Sophora flavescens as the monarch, clearing heat and drying dampness, purging fire and detoxifying; Atractylodes macrocephala,zedoary turmeric, Scutellaria barbata and Hedyotis diffusa as ministers, promoting blood circulation and breaking blood stasis,eliminating accumulation and water, and anti-cancer poison; With Sparganium as the assistant, it can break blood and Qi, eliminate accumulation and relieve pain; Take licorice as the agent to reconcile various drugs; The whole prescription has exquisite flavor and strong power. It plays the functions of clearing away heat and dampness,breaking blood stasis and promoting water, dispersing knot and anticancer.

From the perspective of modern pharmacological research, the composition of Qinggan Huayu granule, baicalein, one of the main components of Scutellaria baicalensis, can inhibit angiogenesis by destroying tumor vascular development [19], and baicalin, another main component, can reduce the expression and activity of VEGF,inhibit tumor angiogenesis, inhibit tumor growth and metastasis [20].Matrine, the main component of Sophora flavescens, can inhibit tumor angiogenesis by inhibiting the proliferation of vascular endothelial cells and down regulating the expression of vascular endothelial growth factor and MMP-9 protein [21]. Studies have shown that Curcumol, the main monomer of zedoary turmeric,can inhibit the expression of VEGF protein and then inhibit tumor angiogenesis [22], and curcuma oil can significantly inhibit the growth and proliferation of liver cancer cells [23]. Different extracts of Scutellaria barbata or different chemical components have certain antitumor activities, which can reduce the content of VEGF and inhibit tumor angiogenesis by reducing the density of platelet endothelial cell adhesion molecule (CD31) [24,25]. Therefore, the author speculates that the effect of Qinggan Huayu granule may be related to inhibiting the increase of peritoneal vascular permeability and peritoneal neovascularization. It is verified by peritoneal pathological examination and OD value detection of ascites supernatant, and the preliminary mechanism of detection of VEGFA and VEGFR2 expression.

VEGF has been proved to be an important mediator of ascites formation and angiogenesis in ascites tumors, and the treatment targeting VEGF can inhibit the production of ascites [26]. VEGFR2 is the main receptor of VEGF and the most important transduction factor of VEGF mediated angiogenesis [27]. Treatment targeting VEGF / VEGFR signaling pathway can inhibit ascites formation.The results showed that the expression of VEGFA and VEGFR2 in the Qinggan Huayu granule group was significantly lower than that in the model group, suggesting that the mechanism of Qinggan Huayu granule may be related to the inhibition of VEGF / VEGFR signal pathway.

Ganfule capsule is a Chinese patent medicine for the treatment of primary liver cancer with liver stasis and spleen deficiency as the main syndrome. It emphasizes eliminating evil and strengthening health, and the efficacy of the formula is comprehensive [28].Qinggan Huayu granule is a method of dispelling dampness, heat,toxin and blood stasis to benefit water. Its medicine tastes less and refined, has strong special effect and reaches the disease directly. In case of emergency, it will cure its target, so as to avoid premature tonic and closing the door and leaving the enemy. The prescription ideas of the two are different. In this study, the two drugs acted on H22 hepatoma ascites tumor mouse model respectively. The results showed that Qinggan Huayu granule had stronger anti ascites effect than Ganfule, which could significantly reduce ascites formation and prolong the survival time of mice.

In this study, H22 hepatoma ascites tumor mouse model was selected, and H22 hepatoma cells were implanted in the abdominal cavity of mice, which can properly simulate the formation of primary hepatoma ascites. The results showed that Qinggan Huayu granule could inhibit the formation of ascites and prolong the survival time of H22 hepatoma ascites tumor mice, and the optimal therapeutic dose was 1.34 g / kg, and the ascites inhibition rate was 25.67%;Through the detection of peritoneal histopathology and OD value of ascites supernatant, it is suggested that Qinggan Huayu granule may play a role in the treatment of malignant ascites by inhibiting the increase of peritoneal neovascularization and the increase of peritoneal vascular permeability. Its mechanism may be related to the inhibition of VEGF / VEGFR pathway.