Explore Acupuncture Combined With Cyclophosphamide On 4T1 Breast Cancer Tumor Growth In Mice And Its Mechanisms Of Influence

Breast cancer is the most common female malignant tumor accounting for about a third of women tumor and7%～10%of whole body all kinds of malignant tumor, which is the second leading cause of cancer death.There are about1.3million people diagnosed with breast cancer and about400000people dying of the disease every year. Breast cancer is mainly treated with operation,while chemotherapy and adiotherapy asadjuvant treatment is extremely important.However, due to the chemotherapy drug resistance and the heterogeneity of the tumor itself or other reasons,there are still a part of the breast cancer recurrence or survival with tumor, how to eliminate the residual tumors and to improve curative effect has been the difficulties of the breast cancer treatment. So there is important significance for breast cancer research.With the development of experimental acupuncture,the acupuncture effect on the immune function of animal experiment is relatively abundant, suggesting acupuncture can improve the immune function, which can infer that it can bring benefits to patients with tumor.In clinical work,we found that tumor can reduce significantly or even disappear with the treatment of surrounding needling and/or combined with chemotherapy,which may improve local tumor immune function to suppress tumor growth.Based on previous literature research and clinical observation in acupuncture treating cancer, this experiment is to verify effect of acupuncture in the treatment of cancer, exploring the mechanism of acupuncture restraining tumor and chemosensitivity.Objective:Starting from the effects of acupuncture on the body’s immune system and tumor microenvironment,to observe tumor inhibitory rate with surround needling combined with chemotherapy,to explore the mechanism of acupuncture anti-tumor, and to provide a new treatment for breast cancer or other refractory tumors.Methods:4T1breast cancer BALB/c mice model was established and randomly divided into4groups,which is respectively model group, medication group, acupuncture group and the needle medicine group.When tumor accumulated up to about5mm,the drug mice were treated with intraperitoneal injection of cyclophosphamide and the needling mice were acupunctured surrounding the tumor.Observe inhibition rate and measure spleen index and thymus index.Flow cytometry was used to detect the expression of tumor cells PD-L1and CD4+CD25+Treg cell percentage.Using HE staining to observe tumor tissue necrosis area, nuclear fission and infiltration.Immunohistochemical method was used to detect the expression of MVD,HSP70and MMP-9in tumor tissue.Results: 1Tumor inhibitory rateThe tumor weight of drug,acupuncture and acupuncture medicine group reduced significantly compared with model group,P<0.05.The acupuncture medicine group showed an obviously higher than drug group and acupuncture group,P<0.05.According to the results above,both CTX and acupuncture have certain inhibitory effect on tumor growth,while the inhibitory effect of acupuncture combined with CTX is stronger,so acupuncture can improve the tumor suppression effects of CTX.2The index of spleen and thymusThe spleen index of model group improved compared with durg group and reduced compared with acupuncture and acupuncture medicine group,P<0.05.The acupuncture showed an obviously higher than acupuncture medicine group in the spleen index and durg group in the thymus index, P<0.05.We can think that CTX can obviously inhibit the mice immunity,while acupuncture can improve immunity. Acupuncture can reduce immunosuppress-ion of CTX in mice.3Observe the tumor cells, necrosis and infiltrationThe range of tomor cells was in disorder.The type and size was different in the tumor cells and nucleus. Pathological karyokinesis was common.We can conclude4T1breast cancer cells with high pleomorphism has an extreme malignancy.While each group had different tumor atypia, model group was maximum and acupuncture group was minimum.The necrosis area of acupuncture group increased significantly compared with drug group, Statistical data of other group has no Significant difference.We can conclude that acupuncture can promote tumor cell necrosis,while there is not Synergistic effect combined with medicine.4The formation of microvascular marked with CD34in the tumor tissueIHC showed that the model group increased significantly compared with the drug group, acupuncture group and acupuncture drug group,while acupuncture drug group showed a drop compared with the drug group and acupuncture group in microvessel positive area and IOD marked by CD34,P<0.05.The OD of needle medicine group was obviously lower than model group, drug group and acupuncture group,P<0.05.No difference was found between drug group with acupuncture group,P>0.05.Accordingly it can be assumed that both acupuncture and CTX have Inhibition of angiogenesis on mouse4T1breast cancer cells, while acupuncture combined CTX show a stronger inhibition of microvascular.5The expression of HSP70in the tumor tissueIHC showed that the model group improved significantly compared with the drug group, acupuncture group and acupuncture drug group in the positive expression area,OD and IOD of HSP70which is not different between drug group and acupuncture group,while acupuncture drug group showed a drop compared with the drug group and acupuncture group in the positive expression area and IOD of HSP70,P<0.05. It can be speculated,acupuncture and CTX on the expression of HSP70in mice have a certain inhibition,but acupuncture combined with CTX can inhibit HSP70expression significantly.6The expression of MMP-9in the tumor tissueIHC showed that the model group enhanced makedly than the drug group,acupuncture group and acupuncture drug group in the positive expression area and IOD of MMP-9which the drug group had a drop compared with acupuncture group and acupuncture drug group, P<0.05.In OD of MMP-9,acupuncture group was lower than model group and acupuncture drug group,meanwhile drug group declined combined model group,P<0.05.It can be thought that acupuncture and CTX on the expression of MMP-9in mice have a certain inhibition,in addition,acupuncture or acupuncture combined CTX shows more inhibition of MMP-9expression significantly.7The expression of PD-L1in the tumor tissueFCM showed that drug group improved significantly compared with model group in the positive rate and expression of PD-L1,P<0.05.Model group was slightly lower than acupuncture group and acupuncture drug group,at the same time, acupuncture drug group showed a bit increase compared acupuncture group, P>0.05.Therefor,we can conclude that CTX improves the expression of PD-L1which plays the tumoral immune suppression effect,while combined acupuncture, to some extent,it can alleviate the inhibitory effect of CTX on tumor local immunity.8The expression of CD4+CD25+Treg cells in the tumor tissueFCM showed that in the percentage of CD4+CD25+Treg cells model group increased significantly than drug group and acupuncture drug group which declined obviously compared acupuncture group,P<0.05.There was no difference between drug group and acupuncture drug group,P>0.05.It can be thought that CTX or CTX combined acupuncture shows an Inhibition of expression in the CD4+CD25+Treg cells.Acupuncture is not found significant Inhibition effect,meanwhile there is no synergistic effect when CTX combines with acupuncture.Acupuncture drug group was lower than model group in expression intensity of CD25,P<0.05,there was no significant difference between other groups. It can be speculated that,acupuncture and CTX has no obvious effect on the local immune of tumor, but acupunctu-re combined CTX can inhibit the tumor local negative immune regulation.9Pathology and FCM data change trend in different experimental intervention period Acupuncture group had a falling in angiogenesis,positive expression of HSP70and PD-L1with the time being, the other three groups trend was not obvious or not uniform.lt suggests acupuncture shows a better long-term efficacy in regulating tumor local microenviro-nment.Conclusions:1In this experiment,acupuncture has certain anti-tumor effect and can play a synergistic effect combined with drug.2Acupuncture can improve the body immunity and reduce the immunesuppressive effect of CTX.3Acupuncture can play a positive regulatory role on local tumor microenvironment, such as reduction of angiogenesis,expression of HSP70and MMP-9.Acupuncture combined with CTX can lower the expression of Treg cell and PD-L in some degree.4With the increase of intervention time, acupuncture group showed a downward trend in tumor angiogenesis,HSP70and PD-L1expression which is speculated that acupuncture has a good long-term effect in improving local tumor microenvironment.If possible,the follow-up experiment will further extend the intervention time,increase the sample size, and observe the survival time of mice.