| Objective:Malignant tumors have become one of the major public health problems that seriously threaten the health of Chinese population in recent years.The incidence and mortality of tumors are on the rise,and the situation of prevention and control is very serious.At present,there is an urgent need to find natural anti-cancer drugs with good anti-tumor effect and little side effects.There are more and more studies on Poria Cocos polysaccharides.Many studies show that Poria cocos polysaccharides have good anti-tumor effects.However,most of them are extracted from the sclerotia of Poria cocos,which has a long culture period and low yield.The extraction of polysaccharides is usually obtained by ultrasound or enzymatic hydrolysis.The extraction of Poria cocos polysaccharide from Poria cocos mycelium fermentation broth by liquid fermentation has the advantages of short period,low cost and high content of water-soluble polysaccharides.There are few studies on its anti-tumor effect.Therefore,this experiment mainly uses liquid fermentation to extract Poria cocos fermentation broth polysaccharide(PFBP)and preliminarily verify its anti-tumor activity in vivo and in vitro.Methods:1.The fermentation broth of Poria cocos mycelium was prepared by fungal liquid medium,and the polysaccharides were extracted by water extraction and ethanol precipitation.The content of polysaccharides was detected by phenol-sulfuric acid method,and the yield of polysaccharides was calculated.2.The H22 mouse hepatoma cells were directly treated by PFBP in vitro.The normal saline was used as blank control and cisplatin was used as positive control.CCK-8 method was used to detect the polysaccharides.The cytotoxicity of PFBP was measured.3.By establishing the H22-bearing Balb/c mice model,the mice were treated with different concentrations of PFBP(50mg/kg,100mg/kg,200mg/kg),saline and cisplatin.The survival time,weight and abdominal circumference of the mice were observed,and the blood smear of tail vein was taken to observe the outside of the experimental mice by Rayleigh-Giemsa staining.Changes of inflammatory cells in peripheral blood.Liver,spleen and thymus of mice were taken and weighed.Eyeball blood of mice was taken to detect IL-2,IFN-y and TNF-? by ELISA kit.Ascites of mice were centrifuged,paraffin sections were made and HE stained to observe the effects on ascites tumor cells and immune cellsResults:1.Poria cocos mycelium fermentation broth was obtained by liquid fermentation of Poria cocos mycelium,and polysaccharide was extracted by water extraction and alcohol precipitation method.The concentration of polysaccharide was 10.1 mg/mL by phenol-sulfuric acid method,and the yield of crude polysaccharide was 4.5%.2.PFBP directly acted on H22 cells in vitro.CCK-8 showed that PFBP could directly inhibit H22 cells in vitro,and present a positive effect.In a time-dose dependent manner,the inhibition rates of the highest concentration(2mg/mL)for 24 hours and 48 hours were 23.12%and 35.7%respectively,which were lower than that of cisplatin for 24 hours and 48 hours(43.34%and 75.96%respectively)(p<0.001).3.The treatment group was given intraperitoneal injection of 50 mg/kg,100 mg/kg and 200 mg/kg of PFBP as gradients,the same as that of cisplatin for 24 hours and 48 hours(p<0.001).When normal saline was used as blank control and cisplatin(20mg/kg)was used as positive control,the results showed that the body weight and abdominal circumference of mice in medium and high concentration group and cisplatin group on the 8th day were significantly smaller than those in blank control group(p<0.01)and the growth rate was slower,and the growth rate of mice in high concentration group was the slowest;4.Two mice in high concentration group of PFBP survived to the 30th day.The mice in saline group died on the 12th day,and all died on the 25th day.One mouse in cisplatin group died on the 20th day,and one mouse in cisplatin group survived on the 30th day.The results showed that the survival time of mice in treatment group and cisplatin group was longer than that in normal saline blank control group.5.There was no significant difference in liver index between treatment group and cisplatin group compared with control group(p>0.05),indicating that there was no significant difference in liver effect between each group.The spleen index of the treatment group and the cisplatin group increased and showed a concentration dependence compared with the thymus index of the blank control group(p<0.001),the spleen index of the cisplatin group was also higher than that of the blank control group,while the thymus index of the cisplatin group had no difference compared with the control group(p>0.05).6.PFBP treatment group in the middle and high concentration phase.Compared with the blank control group,the serum IL-2 level was significantly increased(p<0.05),but there was no difference between the low concentration group and the cisplatin group.The results showed the same trend in serum IFN-y.The serum TNF-? levels in the treatment group and cisplatin group were significantly higher than those in the blank control group(p<0.05),while the serum TNF-? levels in the low concentration group and cisplatin group were significantly higher than those in the blank The levels of these three cytokines in the treatment group were not significantly different from those in the blank control group(p>0.05).7.Peripheral blood smear staining showed that a large number of inflammatory cells infiltrated into peripheral blood in the treatment group,such as neutrophils,lymphocytes,monocytes,and so on.The number of red blood cells was larger and the distribution of red blood cells was tight,while in the saline group and cisplatin group.Cell arrangement is sparse,and inflammatory cells are rarely seen.Ascites tumor cell staining showed that the tumor cells in the treatment group were irregular in shape,small in size,with nuclear edges gathered.Some of them could see nuclear fragmentation.There were a large number of inflammatory cells and immune cells in the ascites,such as neutrophils,lymphocytes and macrophages.In the saline group,the nuclei of the tumor cells were deep-stained and regular in shape.Inflammatory cells were rare.Tumor cells in cisplatin group were irregular in morphology,with incomplete arrangement of nuclei and fewer inflammatory cellsConclusion:1.Polysaccharides were extracted from the fermentation broth of Poria cocos mycelium by water extraction and alcohol precipitation.The concentration of polysaccharides was 10.1 mg/mL,and the yield of crude polysaccharides was 4.5%.2.CCK-8 method was used to detect the cytotoxicity of PFBP on H22 cells in vitro,which confirmed that PFBP had a direct killing effect on H22 cells in vitro.3.The survival time of H22 tumor-bearing mice treated with PFBP was prolonged and the symptoms of ascites were alleviated.4.PFBP could increase spleen and thymus index of H22 tumor-bearing mice,but had no effect on liver index.5.PFBP can promote the secretion of IL-2,IFN-? and TNF-? in serum of H22 tumor-bearing mice,and the infiltration of inflammatory cells in peripheral blood and ascites of mice.PFBP plays an indirect anti-tumor role mainly by stimulating the immune function of the body. |
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