| Objective:The subcutaneous transplanted tumor model of laryngeal squamous cell carcinoma in nude mice was established by in vivo experiments,and the effects of Buzhong Yiqi Recipe on the expression of PI3 K,p-PI3 K,AKT1,p-AKT1,m TOR and p-m TOR proteins in the subcutaneous transplanted tumor tissue of human laryngeal squamous cell carcinoma(LSCC)cells in nude mice were discussed,which partly explained that Buzhong Yiqi Recipe was inhibited by PI3K-AKT1-m TOR pathway.Methods:A total of 40 SPF-grade nude mice were picked,including male and female.The pre-finished Fadu LSCC cell culture medium was adjusted to the experimental standard volume concentration.The nude mice were injected subcutaneously with Fadu cells to establish the LSCC animal model.After successful simulations,the exposed mice were randomly divided into control groups(saline)and the experimental groups with low,middle and high doses of Buzhong Yiqi Decoction.One week after modeling,gastric lavage was started,in which the control group was given normal saline,and the gastric lavage concentrations of low,middle and high dose groups were 0.378g/ml and 0.756 g/ml respectively.1.134g/ml,with a fixed dose of 0.5ml per mouse,once a day for 2 weeks.Check the tumor morphology every day,and observe the drinking,eating and mental state of mice.Tumor growth indicates successful modeling.The maximum major diameter(L)and diameter transversa(S)of the transplanted tumor are measured every three days with vernier caliper,including the volume of the transplanted tumor.After the last dose,the nude mice were put to death,and the tumor body was peeled off in sterile environment,and describe the weight and volume of the graft tumor.The pathological morphological differences to the tumor were recorded using HE staining.Immunoblotting was used to detect PI3 K,p-PI3 K,AKT1,p-AKT1,m TOR and p-m TOR proteins in tumor.Results:(1)After tumor growth,BALB/c mice in each group were in good condition,and no obvious symptoms such as resistance,irritability and abnormal defecation were found.In each group,the tumor volume of the high-dose Buzhong Yiqi Decoction group gradually decreased after 19 days,and the reduction was more significant after 22 days,with statistical difference compared with the control group(P<0.01).However,compared with the control group,the middle dose Buzhong Yiqi decoction group and the low dose Buzhong Yiqi decoction group had no significant difference in the volume of transplanted tumor between groups at each time node of intragastric administration(P>0.05).The reduction of tumor volume in the high-dose group of Zhongyiqitang is obviously related to the increase of gastric perfusion time and the accumulation of drug concentration,which indicates that there is obvious time and dose dependence.On the 22 nd day,the weight of mice in the high dose group was significantly lower than that in the control group,and the difference was statistically significant(P<0.05).However,there was no significant difference in the weight of nude mice between the middle and low dose groups and the control group,and between the middle and low dose groups and the high dose groups(P>0.05).However,after 14 days of intragastric administration,the weight of the tumor in the high-dose group was significantly lower than that in the control group(P<0.05).(2)HE results showed that the capsule of tumor tissue was intact and a few inflammatory cells were found in the capsule.In the low dose group,the capsule of tumor tissue was incomplete,and a few inflammatory cells were found in the capsule.Microscopically,the capsule of two tumor tissues in the middle dose group was relatively complete,and the tumor tissue in the lower left direction was necrotic in a large area,with only some tumor cells remaining in the marginal area.Microscopically,in the high-dose group,the capsule of tumor tissue was intact,and the tumor tissue under the capsule was divided into multiple lobules by fibrous tissue.Most necrotic foci were found in the center of lobules,with different sizes of necrotic foci and mineralization in individual necrotic foci.(3)Western Blot was used to detect the expression of related proteins in different groups: Compared with the control group,the expressions of p-PI3 K,p-AKT and p-m TOR in the high-dose Buzhong Yiqi Decoction group were significantly decreased,and the difference was statistically significant(P<0.05).Compared with the low-dose group,the expression of p-PI3 K and p-m TOR protein in the high-dose group decreased in different degrees,and there was a significant difference(P<0.05).Compared with the control group,the middle and low dose groups and the high and middle dose groups,there was no significant difference in the expression of p-PI3 K,p-AKT and p-m TOR proteins(P>0.05).There was no significant difference in PI3 K,AKT and m TOR protein levels between the control group and the experimental group(P>0.05).Conclusion:1.Buzhong Yiqi recipe can obviously inhibit the growth of subcutaneous transplanted tumor of laryngeal squamous cell carcinoma in nude mice,and it has a positive correlation with the administration concentration;2.PI3 K,AKT and m TOR may be one of the key targets in the progression of laryngeal squamous cell carcinoma,and its mechanism may be that the expression of phosphorylated PI3K-AKT1-m TOR pathway-related proteins is down-regulated in order to inhibit the progression of laryngeal squamous cell carcinoma. |
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