Research On The Effects Of Notch Signaling Pathway Of Acupuncture And Moxibustion On Health Mice Bone Marrow Caused By Cyclophosphamide

Objective: Around the acupuncture and moxibustion improve healthy mice bone marrow suppression after chemotherapy, peripheral blood leukocyte this one theme, further reveals the change rule of acupuncture effect on white blood cells after chemotherapy; To Notch signal pathway research as the breakthrough point, the use of biological chip screening of CTX healthy mice bone marrow hematopoietic cells genetic variations associated with Notch signaling network,immunohistochemical method was applied to further detect differences in gene protein expression, thus further screening acupuncture antagonism in the bone marrow inhibition caused by chemotherapy Notch signaling pathways important signaling molecules, and the use of rt-pcr and Western bolt method to detect these important signaling molecules mRNA transcription level and protein content, from the level of molecular biology to explore the mechanism of action of acupuncture and moxibustion resistance to bone marrow suppression after chemotherapy,chemotherapy after bone marrow hematopoietic function obstacle for acupuncture treatment to provide laboratory basis.Methods:KunMing(KM) mice,SPF, male, 18 ± 2g, 40 mice. In the temperature of 20 ~ 25℃, relative humidity 60% free diet three days inthe experimental environment, after weighting, randomly dividing into the blank group, the model group, the acupuncture group, the moxibustion group and each group of 10 mice; Numbered points cage and picric acid staining. On the basis of new drugs(western medicine)assembly pre-clinical doctrine(the ministry of health of the People’s Republic of China YaoZhengJu) method, CTX mice bone marrow suppression model is set up. Intraperitoneal injection of CTX 100mg/kg/d(in accordance with the weight of 0.02ml/g, match into CTX injection of solution concentration of 5mg/ml), after 24h、48h, inject CTX again. On the basis of cyclophosphamide pharmacokinetic process,pharmacological activity disappeared after 4 hours, model successed(zhongshan school of medicine, etc. By basic pharmacology. People’s medical publishing house, 1979:442.). According to 0.02ml/g dose, the blank group mice, injection of 0.9% with physiological saline injections.According to the chronological sequence of molding, 4h after the success of the model copy, start acupuncture and moxibustion therapy. The blank group and the model group just accompanied by other groups, no treatment. The acupuncture group was fixed mice prone to a homemade wooden boards, selection of beauty filiform needle of Hua Tuo brand(specification: 0.19×10 mm, needle handle: 20 mm), use the tube, stab into 3 mm, retaining needle 6 min, once a day, 5 days in a row. The moxibustion group was fixed mice prone to a homemade wooden boards,light moxa stick(specification:0.4×25cm), 2cm from moxibustion acupoint skin, suspended moxa stick, every point 3 min, 1 times a day, 5days in a row. To observe the effects of acupuncture on leukocyte rule after treatment, and applied to gene expression profile chip bone marrow cells in mice to CTX treatment 5 days after chemotherapy, the differences of Notch signaling pathway genes in preliminary screening, then, the immunohistochemical method was used to detect differences in proteinexpression of genes, thus further screening has significant differences in gene, and then to the significant difference of gene by RT-PCR and Western bolt methods respectively to detect the gene transcription level and protein expression of mRNA.By observing the CTX mice after acupuncture Notch signaling pathway differences in gene expression levels of change, from the aspects of bone marrow cell signal transduction pathway effects of acupuncture and moxibustion improving bone marrow suppression after chemotherapy, improve the mechanism of action of white blood cells.The whole experiment process strictly abide by the national ministry of science and technology published the guidance on treating experimental animals.Results: 1. "experiment 1st " results show that the building before the foundation of the various groups of mice white blood cell count showed no statistically significant difference(P> 0.05). 2 and 3 days after building successful mice peripheral blood leucocyte decrease, than with the blank group, the leukocyte between groups of mice were reduced,statistical significant differences(P<0.05); The acupuncture group,moxibustion group compared with model group, white blood cell count there was no significant difference(P>0.05). 3 days after treatment,acupuncture and moxibustion groups began to rebound, the effect of acupuncture group is better than the moxibustion group, and there are significant differences(P<0.05); In 4~5 days acupuncture group,moxibustion group were significantly elevated WBC, 5 days is normal.2. "experiment 2nd" results show that the application of gene chip for groups of mice bone marrow hematopoietic cells genetic variations associated with Notch signal pathway in preliminary screening, a total of seven different genes screened out: delta1, 2, jag1, jag2 notch1, Notch,numb1, numb2.3. "Experiment 3rd" results showed that treatment for five days, in the blank group, model group,notch2 and jag1 rise, jag2 numb1 and numb2 cut down, and with significant difference(P<0.05); Delta1, notch1 has no statistical significance(P>0.05); In the model group, acupuncture group and moxibustion group of notch2 and jag1 cut, jag2 numb1 and numb2 rise, with significant difference(P< 0.05). Jag2 in than to the acupuncture group, moxibustion group, with significant difference(P<0.05). 7differences in gene expression profile chip select genes in further screening notch, jag1, numb1,numb2 these four genes with significant difference.4. "Experiment 4th" results show, bone marrow cells in health mice after the injection of CTX notch jag1, high notch2 gene expression in the signal path, numb1, numb2 gene significantly lower expression, after acupuncture treatment, Compare to the model group, the acupuncture and the moxibustion group although there was no significant difference, but the acupuncture group, the moxibustion group numb1, numb2 content increased, jag1, notch2 content were reduced.5. " Experiment 5th " results showed that after injection of CTX jag1,notch2 higher protein content, numb1, numb2 protein content decreased,show the numb in the bone marrow cells induced by CTX can cut down the content of protein(numb1, numb2) and lead to notch(jag1, notch2)protein content increased, the notch signaling pathway lose inhibition,abnormal activation, bone marrow suppression. Acupuncture and moxibustion for five days, the acupuncture group, moxibustion group and model group than, jag1, notch2 protein content decreased, with significant difference(P< 0.05); Acupuncture group, moxibustion group and model group, numb2 content increased, and the moxibustion group with significant difference(P< 0.05), the acupuncture group, moxibustion group and model group, numb1 protein quantity change, though there wasno significant difference(P> 0.05), increased, but high in the moxibustion group and acupuncture group. Prompt acupuncture can by increasing numb protein content, lower notch protein content.Conclusion:1. Acupuncture can relieve CTX healthy mice bone marrow suppression, enhance the role of white blood cells, white blood cells and with the change of teacher group for many years to summarize the change rule of consistent: namely in the treatment of 3 days began to rebound, in5 days normal, two methods of acupuncture and moxibustion is no significant difference.2. Through the screening of gene expression profile chip and immunohistochemical, in groups of healthy mice bone marrow hematopoietic cells on the Notch signal pathway, a total of 2, jag1, jag2notch1 selection, Notch, delta1, numb1, numb2 seven genetic variations,including jag1, notch2, numb1, numb2 has significant difference.3. Acupuncture and moxibustion can increase numb1, numb2 mRNA expression, cut jag1, notch2 mRNA expression, can promote the increase of the content of numb1, numb2 protein decreased notch protein content.4. Acupuncture reduce bone marrow suppression caused by chemotherapy of CTX and its possible mechanism is: acupuncture and moxibustion can increase Numb in bone marrow tissue protein(numb1,numb2) content, and then cut the protein content of notch2, jag1, inhibit the excessive activation of Notch signaling pathway, the bone marrow hematopoietic stem/progenitor cells, enhanced cell proliferation and differentiation of cells is reduced, so that the increase in the number of hematopoietic stem/progenitor cells, bone marrow hematopoietic function abate.5. The numb-Notch signal pathway, may be the Notch signaling pathway plays against bone marrow suppression, promote white blood cells, the important way to increase the body’s immune function.