| Hepatocellular carcinoma is clinically common,and it’s also one of the most difficult malignant tumors for treatment.Nowadays,the effective means of treatment of hepatocellular carcinoma is limited.The radically surgical resection of the tumor is the main treatment to the patients with surgical indications.According to different periods and different conditions,it can also be combined with interventional treatment,radiofrequency ablation,immunotherapy,chemotherapy,radiotherapy and Chinese medicine treatment.However,the recurrence rate of hepatocellular carcinoma is high,which is a serious impact on the live quality and the survival to patients.So how to promote postoperative hepatocellular carcinoma hibernation and prevent the recurrence has become a medical hot spot in recent years.With the clinical observation of Chinese medicine treatment to cancer and the gradual deepening of experimental research,we found that traditional Chinese medicine plays an important role in inhibiting tumor cell activity,regulating immunity,improving quality of life and prolonging survival.Jianpi Huayu Decoction is inherited from Shanghai famous doctor professor Qian Bowen,optimized from the famous Chinese medicine professor tutor after the clinical observation and experimental study.with the power of strengthening the resistance and eliminating evil,it aids to the postoperative hepatocellular carcinoma.Jianpi Huayu Decoction can remove cancer evil,also strengthen the resistance to prevent the postoperative recurrence.The experimental study found that Jianpi Huayu Decoction can adjust the immune microenvironment in order to achieve anti-tumor,and promote the therapeutic effect of hibernation,but the specific mechanism is not yet clear.Therefore,it is of great significance to further study the mechanism of Jianpi Huayu Decoction about how to regulate the immunity.Exosomes are small vesicles that widely exist in various cells,tissues and body fluids.They contain a large number of miRNAs such as miR-21,and they are considered as important mediators of cell-cell information transmission.Exosomes play an important role in the whole process of tumors development.miR-21 was found to be a possible carcinogen miRNA.Study found that miR-21 expression and dendritic cell differentiation was negatively correlated.miR-21 reduction or even missing can promote dendritic cell differentiation and stimulate its secretion of IL-12 increasing,in order to activate CD4+T cell differentiation【1】.Therefore,we suppose that Jianpi Huayu Decoction may change the immune microenvironment by affecting the expression of miR-21 in exosomes,so as to promote the dormancy of hepatocellular carcinoma and prevent tumor recurrence.PurposeTo clarify the the expression of miR-21 in the exosomes changing the immune microenvironment in order to affect the postoperative recurrence of hepatocellular carcinoma in vivo experiments。To discuss the effect of Jianpi Huayu Decoction in regulating the immune microenvironment and preventing recurrence of hepatocellular carcinoma by intervening the expression of miR-21 in exosomes.MethodsThe experiment was divided into two parts.The first part:The human hepatocellular carcinoma cell SMCC-7721 was cultured and inoculated on the liver of nude mice to establish a model of orthotopic hepatocellular carcinoma.On the basis of orthotopic hepatocellular carcinoma in nude mice,we established a model of postoperative hepatocellular carcinoma.In order to establish a model of recurrent hepatocellular carcinoma in nude mice,the postoperative hepatocellular carcinoma model was made diet disorder and stimulated by trauma.There were three nude mice model totally.At the period,closely observed and recorded the general situation of nude mice.Combine with anatomical observation and pathological examination to assess tumor formation,dormancy,recurrence.The serum,lung and liver of nude mice were collected and the exosomes were extracted from the serum.The exosomes were identified by electron microscopy and NTA technology.The expression of miR-21 in exosomes was detected by q RT-PCR.Cytometry and ELISA were used to detect the levels of CD4+Tcells and IL-12 in serum,lung and liver respectively.The second part:the same cultivation of human hepatocellular carcinoma cell SMCC-7721.The postoperative hepatocellular carcinoma model were given intragastric administration of Jianpi Huayu Decoction and intraperitoneal injection of pentafluorouracil.Closely observed and recorded the general situation of nude mice.Combine with anatomical observation and pathological examination to assess tumor formation,dormancy,recurrence.The serum,lung and liver of nude mice were collected and the exosomes were extracted from the serum.The exosomes were identified by electron microscopy and NTA technology.The expression of miR-21 in exosomes was detected by q RT-PCR.Cytometry and ELISA were used to detect the levels of CD4+Tcells and IL-12 in serum,lung and liver respectively.Results1、Expression of miR-21 in exosomes affects hepatocellular carcinoma immunity microenvironment.（1）Within 28 days after modeling,the mental status,eating habits and activities of nude mice in each group were normal.No suspicious mass appeared on the body surface of nude mice in each group,and no tumor protruded in liver area.Late stage of hepatocellular carcinoma group,a nude abdomen mild swelling was considered the formation of ascites.（2）The results of pathological examination showed that the tumors resected in the hepatocellular carcinoma group and the hepatocellular carcinoma recurrence group were confirmed to be the foci of tumor,showing the infiltration of tumor cells.No tumor cells were excised from para-tumor tissue in the postoperative hysterical hepatocellular carcinoma group,and the tumors were considered as resected.And the remaining liver,lung,intestine,spleen had no tumor cells,indicating that there were no intrahepatic and distant metastasis in each group.（3）The results of exosome identification showed that most exosomes showed a round shape with small cup-shaped monolayer vesicles.The size of the exosomes mainly concentrated in the range of 133nm,also with peak concentrations at 193nm and 258nm.（4）Negative regulation of miR-21 in exosomes to immune microenvironment in each hepatocellular carcinoma state:The expression of miR-21 in exosomes of four groups was detected by q RT-PCR.The expression of miR-21 in exosomes of hepatocellular carcinoma and hepatocellular carcinoma recurrence group were significantly higher than that of hepatocellular carcinoma postoperative hibernation group（P<0.05）.The levels of CD4+T cells in blood,liver and lung were detected by flow cytometry.The levels of CD4+T cells in hepatocellular carcinoma group were significantly lower than those in normal control group（P<0.05）.In the liver alone,the hepatocellular carcinoma group was significantly lower than the hepatocellular carcinoma postoperative hibernation group（P<0.05）.In the lung alone,the hepatocellular carcinoma recurrence group was significantly lower than the hepatocellular carcinoma postoperative hibernation group（P<0.05）.The levels of IL-12 in blood,liver and lung were detected by ELISA.The levels of IL-12 in hepatocellular carcinoma group and hepatocellular carcinoma recurrence group were significantly lower than those in hepatocellular carcinoma postoperative hibernation group and normal control group（P<0.05）.2、Preventive and therapeutic effect of Jianpi Huayu Decoction on postoperative humor recurrence of hepatocellular carcinoma.（1）During the surgical removal of the tumor to the end of the medication,the mental state,eating and activity of Jianpi Huayu Decoction group are normal.21 days after the administration to the end of the medication,five nude mice in the hepatocellular carcinoma postoperative hibernation group showed mildly abdominal fullness and suspicious ascites formation,One nude mouse appeared emaciated,while two nude mice showed both mildly abdominal fullness and emaciation at the same time.Five nude mice in the pentafluorouracil group showed weight loss while one nude mouse was with mildly abdominal fullness and weight loss at the same time.Nude mice of Jianpi Huayu Decoction group did not appear abdominal fullness,weight loss and other adverse conditions.No suspicious mass appeared on the body surface of nude mice in each group,and no tumor protruded in liver area.The incidence of cachexia symptoms in three groups of nude mice were compared.The results showed that the symptoms of early cachexia in Jianpi Huayu Decoction group were significantly less than those in the postoperative hibernation and pentafluorouracil groups（P<0.05）.（2）Histopathological examination showed that 13,9 and 8 nude mice,respectively of the postoperative hibernation group、Jianpi Huayu Decoction group and pentafluorouracil group,had recurrence of hepatocellular carcinoma after operation,showing the infiltration of tumor cells.No tumor cells were observed in the adjacent tissues of nude mice without suspicious recurrence in each group,which could be considered as tumor without recurring.And the remaining liver,lung,intestine,spleen had no tumor cells,indicating that there were no intrahepatic and distant metastasis in each group.（3）The results of exosomes identification showed that most of the exosomes showed a round shape and a small number of vesicles with a cup-shaped monolayer.The size of the exosomes was mainly concentrated in the range of 161 nm,also with the peak of the concentration at 221 nm.（4）Effect of Jianpi Huayu Decoction on the expression of miR-21 in exosomes to the immune microenvironment:The expression of miR-21 in exosomes of three groups was detected by q RT-PCR.The expression of miR-21 in the exosomes of Jianpi Huayu Decoction group was lower than that in the postoperative hibernation and pentafluorouracil groups（P<0.05）.The levels of CD4+T cells in blood,liver and lung were detected by flow cytometry.At the blood,the levels of CD4+T cells in the Jianpi Huayu Decoction group and the pentafluorouracil group were significantly higher than those in the postoperative hibernation group（P<0.05）.At the liver,the level of Jianpi Huayu Decoction group was higher than that of the postoperative hibernation group and pentafluorouracil group（P<0.05）.At the lung,the levels of pentafluorouracil group were higher than that of Jianpi Huayu Decoction group and postoperative hibernation group（P<0.05）.The levels of IL-12 in blood,liver and lung were detected by ELISA.The level of IL-12 in the pentafluorouracil group was higher than that in the postoperative hibernation group and the Jianpi Huayu Decoction group（P<0.05）.ConclusionUnder the condition of hepatocellular carcinoma,miR-21 in exosomes has a negative regulatory effect on tumor immune microenvironment,and Jianpi Huayu Decoction can improve the immune microenvironment and enhance immunity by inhibiting the expression of miR-21 in exosomes.So Jianpi Huayu Decoction can also prevent postoperative recurrence of hepatocellular carcinoma and improve quality of life to the extent possible. |
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