| ObjectiveThis study discusses the inhibition effect of Ganji Fang which disperses stagnated liver qi and strengthening spleen and promoting blood circulation for removing blood stasis on the weight and volume of tumor of the human hepatoma HepG2 bearing mice which are in the type of liver-qi stagnation and spleen deficiency.In this study,we explore the effect of Ganji Fang on liver index and spleen index of mice bearing tumor and the expression of Bax,Bcl-2and Caspase-3 apoptosis related proteins in tumor tissues.In clinical selection,we choose liver cancer patients who are in the type of liver-Qi stagnation and spleen deficiency.We access the effection of Ganji Fang on clinical symptom score,KPS score,classification of liver function,and Immunological indexs such as CD3+?CD4+?CD8+?mHLADR?CD64+and so on.In this study,we preliminarily explored the clinical efficacy and its hypothesized mechanism of action of Ganji Fang by animal experiment and clinical studies.Method1.Animal experimental study1.1 Animal model establishment and grouping32 BALB/c nude mice were randomly divided into model group?16 rats?and control group?16 rats?,model group with chronic restraint stress and excess fatigue and improper diet method composite stress models were made for 28 days,the modeling method refered[1]:nude mouse limit 3 hours in a shackle 10ml bound syringe in the morning at 8:00,and at 2:00 in the afternoon are filled with warm water?25±1°C?large plastic bucket swimming in 10min.Every other day,feeding?fasting every other day,feeding every other day?to high fat and high cholesterol diet.The control group did not intervene,given ordinary feed and free to eat and drink.The changes of general conditions,body weight,food intake and tail suspended time of nude mice were observed.The results showed that the nude mice model of liver depression and spleen deficiency was successfully established.Experimental groups:model group mice were randomly divided into Model Ganji Fang Group?MG?,Model Normal Saline Group?MN?,control group were randomly divided into Control Ganji Fang Group?CG?,Control Normal Saline Group?CN?,8 rats in each group,four groups were established in human hepatocellular carcinoma cell line HepG2 transplanted tumor the nude mice model,MG group and CG group treated Ganji Fang gavage,which were calculated equivalent dosage was 28.6g/kg,MN group and CN group were given an equal volume of saline,1 times daily for 30 days.1.2 Observation of general situation,weight and food intake in all groups of tumor bearing miceGeneral condition:daily observation and record of mental status,posture,fur color and activity,nude mucosal color,ear color and feces in nude mice.Weight change:weighing the body weight of nude mice in each group by electronic balance at 8:00 a.m.and plotting the weight growth curve.Change in food intake:at 8:30 a.m.observe the intake of 24h in nude mice.The intake of nude mice per cage was recorded and divided by the number of nude mice,and the average intake of each nude mice was recorded and recorded.The cage was checked before the experiment to ensure the stability of the feed box and no gaps in the material.The feed was added at the same time every day.After 24h,the residual feed was weighed,and the continuous observation was 28 days.Food intake?g?=feed?g?-residual amount?g?.1.3 Evaluation of mass and volume of tumor bearing miceEvery 5 days,tumor size,mobility and hardness were observed and recorded.The measurement method was:the length diameter?a?,the width diameter?b?and the height?c?of the tumor were measured by vernier caliper in vitro,and the volume calculation formula was V=pion abc/6.The end of the experiment,after weighing the nude mice were killed,the tumor was removed,remove blood,fat and other non tumor tissue,electronic scale tumor weight,inhibition rate according to the formula=tumor?control group average tumor weight of treatment group the average tumor weight?/tumor weight of control group x100%,the inhibition rate was calculated.The size of the tumor was measured with a vernier caliper and volume was calculated according to the volume formula,and the volume growth curve of the tumor was plotted.1.4 Calculation of liver index and spleen index in each group of tumor bearing miceAt the end of the experiment,the spleen and liver were removed from the liver and the weight and weight of the liver and spleen were collected by electronic balance scale.According to the organ index,the=10x organ weight?g?/body weight?g?was calculated,and the liver index and spleen index were calculated.1.5 Detection of Bax,Bcl-2 and Caspase-3 apoptosis related proteinsThe effect of Western blot on the expression of Bax,Bcl-2 and Caspase-3protein was detected.At the end of the experiment,the tumor was removed by asepsis.The tissue was cut into small fragments.The homogenate was homogenized,and the total protein of each group was extracted by RIPA lysate.The BCA protein quantitative kit was used for quantitative analysis.Cut 10%separation gel,SDS-PAGE gel electrophoresis of 5%concentrated gum.Each hole by adding 60 g protein,80V constant,for bromophenol blue into the separation gel,the voltage of the mentioned 120V,until the bromophenol blue reached the separation of rubber.The 300mA constant current wet turn changes the protein from the gel to the PVDF membrane.5%BSA 2H TBST closed at room temperature,after washing the PVDF membrane into the antibody solution dilution of 1:1000,4C were incubated overnight,after washing,adding1:2000 two anti dilution solution 2H at room temperature,after washing,the enhanced chemiluminescence liquid?ECL?coated on the surface of the PVDF film,in the darkroom exposure X,optical film clip in developing,fixing.Western blot the use of optical density scanning,using Quantity One software to analyze the gray value compared to beta-actin for reference,the relative expression of Bax,Bcl-2 and Caspase-3 were calculated.2.Clinical study2.1 Inclusion,exclusion criteria and grouping2.1.1 Inclusion criteria:by clinical or pathological diagnosis of primary liver cancer patients,and the TCM syndrome of liver and spleen in patients with spleen deficiency;the age of 18-75 years;the book survived over 3 months;Karnofsky=50 patients;the patients voluntarily accept the clinical stage of experiment other effective anti-tumor therapy;the subjects of good compliance.2.1.2 Exclusion criteria:Patients with liver metastases or with two or more than two kinds of malignant tumor;the near January had at least underwent radical surgery,chemotherapy,radiotherapy,and one of the target to the minimally invasive or TACE effective antitumor therapy;the patients with systemic infection and severe acute and chronic infections have not been accused of the merger wound healing;combined with the important function of serious heart,brain,lung,kidney and other organs have not serious complication in or.2.1.3 Group:selected the First Affiliated Hospital of Guangzhou University of Chinese Medicine from March 1,2017 to March 1,2018 in outpatient and inpatient department of liver stagnation and spleen deficiency syndrome of primary liver cancer patients were 40 cases,using the method of prospective,randomized,controlled,divided into the treatment group?20 cases?and control group?20 cases?,treatment group was treated with Ganji Decoction orally.One day,a total of 30 agents,soil for 2 days,control group was given the decoction of Chinese medicine,the two groups were given medical education,psychological counseling.2.2 Clinical symptom integral scaleBefore and after treatment?30 soil 2 days?,the clinical symptom score scale was evaluated for the treatment group and the control group.Primary liver cancer symptom rating standard reference State Drug Administration organized the compilation of"Chinese medicine clinical research guiding principles?Trial?"guiding principles of clinical research on new drugs of traditional Chinese medicine for the treatment of primary liver cancer?Appendix Part 1:primary liver cancer symptom classification quantization table?.The standard of TCM syndrome diagnosis of liver stagnation and spleen deficiency syndrome of the State Drug Administration organized the compilation of"Chinese medicine clinical research guiding principles?Trial?"guiding principles of clinical research on new drugs of traditional Chinese medicine treatment of liver stagnation and spleen deficiency syndrome part?Appendix2:liver stagnation and spleen deficiency symptoms grading quantitative table?.According to the above classification quantization table main observation of TCM Ganji Decoction on clinical symptoms of liver stagnation and spleen deficiency syndrome such as flank pain,depression,anorexia,abdominal distension,belching,lump,stool etc.to improve the situation.According to the proportions of the severity of each symptom?the specific scoring standard is shown in Appendix 3?.2.3 KPS scoring standardBefore and after treatment?30 days,2 days?,the KPS score of the treatment group and the control group was evaluated,and the specific scoring criteria were evaluated according to the international standard Karnofsky of physical fitness for cancer patients?Appendix 4?.2.4 Classification of liver functionBefore and after treatment?30 days,2 days?,the liver function was evaluated in the treatment group and the control group.The specific assessment method was carried out according to the Child-Pugh scoring system?Appendix5?.2.5 Related immune indexesPeripheral blood T lymphocyte subsets?CD3+,CD4+,CD8+,CD4+/CD8+?,immune related indicators and bacterial infection two items?mHLADR,CD64+,CD14+?were assessed by flow cytometry before and after treatment?30 days,2 days?respectively in two groups.3.Statistical methodSPSS21.0 was used for statistical analysis,and the level of alpha=0.05was tested.Animal experiment:the quantitative data between two groups were compared by two independent sample t test,and the comparison between multiple groups was compared by ANOVA.The LSD method was used in 22 comparisons between groups.The quantitative data were described by means of mean standard deviation.Clinical study:the comparison of quantitative data between the two groups was compared with two independent sample t tests,and the quantitative data were described with mean standard deviation.The comparison of the data between the groups was compared to the chi square test.The description of the classification treatment was frequency and percentage.In the treatment group and the control group,the paired t test or the rank sum test of the two related samples were used before and after the treatment.Result1.Animal experimental study1.1 The effect of hepatoma on the general condition,weight and food intake of the tumor bearing miceBefore administration,the body weight of group MG and group MN was significantly lower than that of group CG and group CN,and the general situation was poor,which was related to the early stage of liver depression and spleen deficiency.At the beginning of the study?before 7D?nude mice showed normal diet,behavior,and the latter?10d?with individual MN nude mice with the rapid growth of the volume of the tumor,eating,drinking,activity and reaction ability gradually becomes poor,emaciation,arching,piloerection,sluggishness,drowsiness,diarrhea and other symptoms,a cachexia shape,while in MG group,the tumor volume growth is relatively slow and MN group,diet,behavior,reaction ability,nutritional status,tumor volume in CG group grew more slowly and the diet,behavior,reaction ability,good nutritional status,tumor volume in group CN than in group MG and group MN increased slowly.There was no natural death of the nude mice in the whole experiment.1.2The effect of hepatoma on the mass and volume of the tumorThe tumor weight of the tumor-bearing mice in the MG group and MN group was significantly larger than that of the CG group and the CN group.The tumor weight of four groups of tumor-bearing mice?`x±s,g?were ranked as MN group?2.65±0.34?,MG group?2.23±0.21?,CN group?1.54±0.13?,and CG group?1.17±0.16?.The difference between four groups was statistically significant.From the volume growth curve of the tumor,the size of the tumor size was:group>MG,group>CN,group>CN,group>CN,group>CG,and liver depression and spleen deficiency model group with larger tumor volume.According to the growth curve of tumor volume,after fifteenth days,the volume of tumor in group MG was significantly smaller than that in group MN?P<0.05?.After twentieth days,the volume of tumor in group CG was significantly smaller than that in group CN?P<0.05?,and the decrease between MG group and MN group was more obvious than that in CG group and CN group.1.3 Effect of Ganji prescription on liver index and spleen index in mice bearing tumorThe spleen index ranking:CG group>CN group>MG group>MN group,Ganji Fang intervention group CG spleen index increased compared with the CN group,MG group,the spleen index increased compared with the MN group,there was no significant difference between the four groups?P>0.05?,suggesting that the model of liver stagnation and spleen deficiency model group CG group,CN group and the spleen the index is lower than that of MG group and MN group;the liver index ranked CG group>CN group>MG group>MN group,Ganji Fang intervention group CG liver index increased compared with the CN group,MG group,the liver index increased compared with the MN group,and there was a significant difference between CG group and CN group?P<0.05?,no significant differences in other among the groups.1.4 The effect of Ganji prescription on the expression of Bax,Bcl-2 and Caspase-3 apoptosis proteinWestborn blot was used to detect the expression level of Bcl-2,Bax and Caspase-3 protein.Westborn blot results showed that,compared with MG group and MN group,CG group compared with CN group,the expression level of Bcl-2protein decreased,increased the expression level of Caspase-3 and Bax protein,suggesting that liver accumulation can increase the expression of Bax protein and down regulating the expression of Bcl-2,Bax increased/bcl-2 ratio,and increase the expression of Caspase-3.2.Clinical research2.1 General situation of patientsThere was no significant difference in general conditions among groups,such as age,sex,tumor shape,diagnosis type,pathological type,tumor location,history of hepatitis cirrhosis,clinical stage,preoperative operation and intervention history.2.2 Change of clinical symptom integralAfter treatment,the clinical symptom scores of the two groups were compared:the total score of the treatment group before and after treatment was statistically significant?P<0.001?.There was no statistical difference in the total score of the control group before and after treatment?P>0.05?.After treatment,the total effective rate of the treatment group was significantly different from that of the control group?P<0.05?.It shows that the two groups are comparable.Comparison of two groups of patients with clinical symptoms of liver stagnation and spleen deficiency after treatment:group before and after comparison of the results,flank pain,depression,anorexia and other indicators in the treatment group before and after treatment were statistically significant?P<0.05?.There was no significant difference between the symptoms of the control group before and after the treatment?P>0.05?.In addition,after treatment,the comparison results between treatment group and control group.The treatment group and the control group between the hypochondrium distending pain,depression,anorexia and other indicators also had no significant difference,wiry or fine,belching,lump,tongue fur,loose stools,abdominal distension,emaciation and hypodynamia lag other indicators in the treatment group and control group differences.2.3 KPS score changeAfter treatment,the KPS scores of the two groups were compared:after the treatment,the scores of the two groups were all improved,but the improvement was not significant?P>0.05?.2.4 Grading changes of liver functionAfter treatment,the liver function classification of the two groups was compared:the difference of liver function before and after treatment was not statistically significant in the treatment group.After treatment,there was no significant difference in liver function between the treatment group and the control group.2.5 Changes of immune related indexesIn this study,20 cases of liver cancer treated by liver stagnation and spleen deficiency type of qyhj decoction treatment group patients after peripheral blood T lymphocyte subsets and 20 cases of the control group comparison:after treatment of peripheral blood CD8+T cells decreased?P<0.05?,indicated that Ganji decoction can make CD8+T the percentage of higher levels of liver spleen deficiency liver reduced;peripheral blood CD3+,CD4+and T cells in the treatment group increased?P<0.05?;the patients in the treatment group CD4+/CD8+increased significantly?P<0.05?;the two groups,the treatment group and the control group CD64 index were significantly increased,compared with no statistical significance before and after treatment,no significant difference between the two groups?P>0.05?;the treatment group,CD3+/mHLADR+was elevated,the difference was statistically significant?P<0.05?;the two group of mHLADR+/CD14 were higher than that before treatment,the difference was not statistically significant?P>0.05?.ConclusionAnimal experiment results show that:the Ganji Fang has a certain inhibitory effect on the quality and volume of human liver cancer HepG2 nude mice subcutaneously transplanted tumor,and at the same time,it can improve food consumption and slow down the weight loss of the tumor bearing mice,and improve the liver index.The expression of Bax protein,the ratio of Bax/bcl-2,and the expression of Caspase-3 protein were up-regulated by Ganji Fang,and Ganji Fang can down regulate the expression of Bcl-2 protein.The clinical study shows that Ganji Fang can improve the liver stagnation and spleen deficiency type of hepatocellular carcinoma patients with flank pain,depression,anorexia and other major clinical symptoms,reduce the clinical symptoms scores;and at the same time the Ganji Fang can incresase peripheral blood CD8+,CD4+,CD3+decreased,CD4+/CD8+ratio,CD3+/mHLADR+ratio.Preliminary animal experiments and clinical studies show that the Ganji Fang has certain protective effects on liver and tumor growth control,and can improve the cellular immune function of liver cancer patients,inhibit the growth of tumor and induce the apoptosis of hepatoma cells. |
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