| Breast cancer is one of the most commonly diagnosed malignancies in women,and is increasing year by year,occupying the first place for female tumors.Up till the present moment,conventional treatments,such as chemotherapy,surgery,endocrine therapy and targeted therapy,are the main treatment choice for breast cancer patients.At present,oral herbal medicine is the main Traditional Chinese Medicine treatment for breast cancer.It had been recognized for certain efficacy.And there are rarely reports and researches about treatments for breast cancer using external treatments of Traditional Chinese Medicine,therefore exploring the mechanism of traditional acupuncture therapy for breast cancer will provide theoretical basis for the application of external treatments of Traditional Chinese Medicine in the treatment of tumor,and this alsowill play an important role in the development of traditional Chinese medicineoncology.In our previous study,we selected 4T1 mouse breast cancer(triple negative)as the object of observation,observingthe antitumor effect of peritumoral acupuncture combined with cyclophosphamide.In this study,we found that peritumoral acupuncture alone has certain antitumor effect,mainly in inhibition of tumor angiogenesis and adjusting local tumor microenvironments.Based on results of previo us research and pre-experiment,we explored a more suitable way and parameter of peritumoral electric acupuncture.We also selected bevacizumab,a recognized targeting drug which inhibits VEGF,as positive control group.We chose ELISA and immunohistochemistry methods to observe the differences between peritumoral electric acupuncture and bevacizumab in inhibiting angiogenesis,and explored the possible mechanism of peritumoral electric acupuncture,which was of great significance to reveal the mechanism of electroacupuncture in antitumor and inhibiting angiogenesis.Objective:To observe the inhibitory effect of peritumoral electric acupuncture on angiogenesis,and to explore the possible mechanism of anti-angiogenesis with peritumoral electric acupuncture by ELISA and immunohistochemistry.Methods:After establishing a model of 4T1 mice transplanted breast cancer,the mice were randomly divided into 5 groups:bland group(KB group),model group(MX group),peritumoral electric acupuncture group(ZC group),bevacizumab group(BF group),peritumoral electric acupuncture and bevacizumab group(ZB group).When the maximum diameter of tumor reached 5mm,we started the experimental intervention Since the beginning of the intervention,every 7 days is a intervention cycle for a total of 3 cycles,ZC group was given peritumoral electric acupuncture every day,BF group was gieven peritumoral subcutaneous injections on the the first day of each cycle,ZB group was given bothperitumoral electricacupuncture and injections at the same time.We collected tissues sampling respectively on 7th,14th,21st days of the whole intervention.We alsomeasured and recordedmice weight and tumor sizedaily,observed the general state of mice and counted tumor inhibition rate,spleen index,thymus index.Concentration of serum VEGF,NGF,TNF-α was detectedby ELISA method,and expression of CD34、VEGFR、NGFR、MMP-2、TIMP-2、MMP-9、NRP-1、Sema3a、DLL4、Inte grin β5、Angl in the tumor tissue weretested by immunohistochemistry at each time point.Results:The results showed that the successfull rate of model was 100%,the survival rate of mice was 100%,and the weight of tumor-bearing mice in each group had some volatility.The tumor inhibition rate of ZC groupincreasedgradually up to 42.71%,while the BF group decreased,ZB group fluctuated.As to overall immunity,acupuncture can reduce the spleen index in a certain extent,and it showed statistical difference gradually with the extension of treatment time.Both acupuncture and bevacizumab can reduce tissue expression of CD34,the trends of changes in each groups were consistent,microvessel density of each time point was MX group>ZC group>BF group>ZB group.The level of serum VEGF in both ZC group and ZB group VEGF decreased earlier than BF group,and the long-term inhibition of serum VEGF expression in ZC group was more stable than the other groups(the serum VEGF level of 14th and 21st days unchanged basically).Opposite to the overall decline or stability trend,expression of serum VEGF in ZB group rose after the first drop.The expression of EGFR in tumor tissue between each groupshas no statistically difference,(P>0.05).Acupuncture has no obvious difference between groups on expression of serum NGF at different time points,but acupuncture could increase the expression of tissue NGFR significantly.The amount relationship of NGFR expression at each time point was ZC group>MX group>BF group/ZB group,the difference between ZC group and the other groups was statistically significant(P<0.05)on the 21st day.Acupuncture can also significantly reduce the expression of MMP-2 and increase the expression of TIMP-2,regulating the ratio of MMP-2/TIMP-2,but it has no significant effect on the expression of MMP-9 with anystatistical difference.The expression of NRP-1 in ZC group decreased firstly(lower than the model group),which was lower than that of MX group almost in the whole process of intervention.While NRP-1 expression of BF group and ZB group at different time points were higher than that of MX group and ZC group.Change of Sema3a was similar to that of VEGF and NRP-1 in MX group.Acupuncture can inhibit the expression of Sema3a at a certain extent,and it was the most obviouson the 21st day with a significant difference with MX group.Both acupuncture and bevacizumab could reduce expression of TNF-α at a certain extent,but there was no significant difference between them.Acupuncture also could inhibit the expression of DLL4 and Integrin β5,but its effect was relatively weak.Conclusion:As to the tumor inhibition rate,ZC group were significantly higher than that of BF group and ZB group at different time with a better long-term effect.ZB group did not appear cumulative effects by using acupuncture and bevacizumab at the same time.For the overall immunization of mice,acupuncture could regulate mice immune state and inhibit the proliferation of lymphocytes in a certain degree,and the longer the time,the more obvious differences between groups.Acupuncture could inhibit angiogenesis,but its strength was less than bevacizumab.Effect of acupuncture on serum VEGF came earlier than bevacizumab.Two kinds of intervention model,bevacizumab and acupuncture,had no synergistic effect and no significant difference on the expression level of EGFR and the concentration of serum NGF at each time point.Acupuncture could significantly increase the expression of tissue NGFR,smiliar to the trend of tumorinhibition rate.This suggested that acupuncture might inhibit tumor by preventing the expression of NGFR.Acupuncture could inhibit the expression of MMP-2 and increase the expression of TIMP-2,regulating the ratio of MMP-2/TIMP-2,which could reduce the degradation of extracellular matrix,inhibit angiogenesis and tumor metastasis.Acupuncture could also reduce the expression of NRP-1,which could inhibit angiogenesis of VEGF.While bevacizumab induced increasing expression of VEGF when it inhibited expression of NRP-1.As NRP-1 could enhance the combination of VEGF165 and VEGFR-2 as a coreceptor,which may was the cause of poor long-term antitumor effect of bevacizumab.Acupuncture can reduce the expression of Sema3a.Based on the above data,it was concluded that acupuncture may regulate the binding of Sema3a-VEGF-NRP-1 and inhibit the growth of tumor.Both acupunc ture and be vac izumab could reducesecretion of pro inflammatory cytokine TNF-α at a certain extent,which led to the indirect effect of regulating tumor growth Acupuncture could change tumor microenvironment into the disadvantage of angiogenesis by inhibiting the expression of DLL4 and Integrin β5 at a certain. |
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