Study On The Sympathetic Regulatory Mechanism By Which Electroacupuncture Alleviates Chemotherapy-Induced Myelosuppression In NSCLC Mice

ObjectiveAmong the various treatment options for lung cancer,chemotherapy is an indispensable first-line treatment strategy,but its many toxic side effects,such as bone marrow suppression,seriously affect the prognosis of lung cancer patients.Acupuncture is a commonly used complementary alternative therapy with more definite clinical efficacy in improving chemotherapy-induced peripheral nerve damage and leucopenia,a previous study identified the modulatory effect of electroacupuncture on bone marrow haematopoietic stem progenitor cells in a mouse model of cisplatin anaemia,and further explored the mediating role of pituitary adenylate cyclase-activating peptide（PACAP）and its receptor PAC1 in the improvement of bone marrow haematopoietic stem progenitor cell subpopulations in chemotherapy treated mice by electroacupuncture on this platform.Based on this effect,this study was conducted in a mouse model of non-small cell lung cancer（Lewis）to elucidate whether electroacupuncture has the same ameliorating effect on bone marrow suppression after chemotherapy in non-small cell lung cancer mice,and the mechanism of sympathetic nerve-mediated electroacupuncture in regulating the hematopoietic microenvironment in mice with bone marrow suppression,so as to provide a scientific basis for the clinical application of electroacupuncture in the effective treatment of bone marrow suppression after chemotherapy in non-small cell lung cancer patients.MethodsExperiment 1 Study on bone marrow suppression of lung cancer mice after chemotherapy by electroacupunctureLewis lung cancer-bearing mice were randomly divided into tumor group（T group）,tumor+chemotherapy group（TC group）and tumor+chemotherapy+electroacupuncture group（TCE group）.The dose of chemotherapeutic agent cisplatin was selected as 3mg/kg twice a week for 2 weeks,and low-intensity electroacupuncture stimulation（0.76m A,5/25Hz,sparse and dense waves）was given to the bilateral ST36 and SP6 three times a week for 2weeks.Bone marrow haematopoietic stem progenitor cells（HSPCs）and their subpopulations（long-term haematopoietic stem cells（LT-HSCs）,short-term haematopoietic stem cells（ST-HSCs）,multipotent progenitor cells（MPPs）,lymphoid progenitor cells（CLPs）,myeloid progenitor cells（CMPs）,granulocyte-macrophage progenitor cells（GMPs）,megakaryocyte/erythroid progenitor cells（MEPs））,and further the number of bone marrow cell cycles（G₀/G₁,S,G₂/M,G₂/M+S）were detected by flow cytometry to observe the effect of electrophoresis on the hematopoietic stem cell subpopulation in mice treated with chemotherapy for non-small cell lung cancer.Experiment 2 Effect of electroacupuncture on bone marrow mesenchymal stem cells in mice receiving chemotherapy for lung cancerIn experiment 1,electroacupuncture could effectively improve bone marrow suppression after chemotherapy in Lewis lung cancer mice,and electroacupuncture could enhance the tumor suppressive effect of chemotherapy drugs,but the tumor suppressive effect of chemotherapy drugs alone was not obvious.In this experiment,5mg/kg chemotherapy drugs were used twice a week for 2 weeks,and the tumor suppressive effect of electroacupuncture combined with chemotherapy was repeatedly verified.The tibia bones of mice were embedded and sectioned,and the distribution and number of bone marrow mesenchymal stem cells（BMSCs）were observed by immunofluorescence staining of bone marrow nestin to clarify the regulatory effect of electroacupuncture on BMSCs in Lewis lung cancer mice treated with chemotherapy.Experiment 3 Study on the role of sympathetic nerve in electroacupuncture in regulating hematopoietic microenvironmentLewis lung cancer-bearing mice were established and randomly divided into tumor group（T group）,tumor+chemotherapy group（TC group）,tumor+chemotherapy+electroacupuncture group（TCE group）,tumor+chemotherapy+6-OHDA antagonist group（TC6 group）,tumor+chemotherapy+electroacupuncture+6-OHDA antagonist group（TCE6 group）tumor+chemotherapy+PAC1 agonist group（TCP）,tumor+chemotherapy+electroacupuncture+PAC1 antagonist group（TCEP）,mice in TC6 and TCE6 groups were injected intraperitoneally with the sympathetic antagonist hexahydroxy dopamine（6-OHDA）on the day 4 and day 5 before tumor loading,the chemotherapeutic agent cisplatin at a dose of5 mg/kg twice a week for 2 weeks,and electroacupuncture was applied to the bilateral ST36and SP6（same parameters as in Experiment 1）twice a week for 2 weeks.On the day of electroacupuncture treatment,PAC1 antagonist was given to mice in the TCEP group and PAC1 agonist was given to mice in the TCP group,and the material was taken on day 21.The number of bone marrow haematopoietic stem progenitor cells（HSPCs）and their subpopulations（LT-HSCs,ST-HSCs,MPPs,CLPs,CMPs,GMPs,MEPs）and the number of bone marrow cell cycles（G₀/G₁,S,G₂/M,G₂/M+S）were measured using flow cytometry to observe the effect of sympathetic and PACAP/PAC1 receptors on electroacupuncture to improve proliferative function of bone marrow hematopoietic stem progenitor cells.The mouse tibiae were embedded and sectioned after sampling,and nestin immunofluorescence staining was used to observe whether sympathetic nerves and PACAP/PAC1 receptors mediated the proliferative function of bone marrow mesenchymal stem cells improved by electroacupuncture.Results1.Electroacupuncture can increase the number of CLPs,CMPs and MPPs in mice receiving chemotherapy for lung cancer,increase the number of G₂/M+S bone marrow cells,and enhance the antitumor effect of chemotherapy drugs in Lewis lung cancer mice.The results of bone marrow stem progenitor cell population analysis on day 21 suggested that,compared with the tumor group,the bone marrow HSPCs（P<0.05）,LT-HSCs（P<0.01）,ST-HSCs（P<0.05）,MPPs（P<0.05）,CLPs（P<0.05）,and GMPs（P<0.05）of Lewis lung cancer mice treated with chemotherapy The number of bone marrow cell cycle ratios did not change significantly after chemotherapy compared with the tumor group;electroacupuncture increased the number of bone marrow CLPs（P<0.01）,CMPs（P<0.01）and MPPs（P<0.05）in Lewis lung cancer mice treated with chemotherapy.Meanwhile,electroacupuncture increased the number of bone marrow cells in the G₂/M+S stage（P<0.05）of Lewis lung cancer chemotherapy mice,suggesting that electroacupuncture could improve the proliferation of bone marrow hematopoietic cells in Lewis lung cancer chemotherapy mice and ameliorate bone marrow suppression after chemotherapy.Compared with tumor group,tumor volume of mice in electroacupuncture group decreased on day 21（P<0.05）,suggesting that electroacupuncture combined with chemotherapy can inhibit the tumor growth of Lewis lung cancer mice2.Electroacupuncture increases the number of nestin⁺bone marrow mesenchymal stem cells（BMSCs）in mice treated with lung cancer chemotherapy.Compared with the tumor group,the tumor volume of mice in the chemotherapy group and the electroacupuncture group decreased on day 14,17 and 21（P<0.01）,suggesting that electroacupuncture combined with chemotherapy can inhibit the tumor growth of Lewis lung cancer mice.On the 21st day,the expression of nestin⁺MSCs was increased in Lewis lung cancer mice after electroacupuncture intervention compared with the tumor group（P<0.001）;the expression of bone marrow MSCs was also increased in the electroacupuncture group compared with the chemotherapy group（P<0.001）,suggesting that electroacupuncture can upregulate the expression of bone marrow MSCs in Lewis lung cancer chemotherapy mice.3.Sympathetic and PAC1 receptor-mediated effects of electroacupuncture to increase the number of multiple hematopoietic stem progenitor cell subpopulations in the bone marrow of mice treated with lung cancer chemotherapy and PAC1 antagonist-mediated effects of electroacupuncture to improve the expression of nestin⁺mesenchymal stem cells in mice treated with lung cancer chemotherapy.On day 21,the subsets of bone marrow hematopoietic stem progenitor cells and bone marrow cell cycle were analyzed by flow cytometry.The results revealed that the number of bone marrow GMPs（P<0.001）and the number of S-phase（P<0.05）and G₂/M+S-phase（P<0.05）bone marrow cells were significantly reduced in mice in the chemotherapy group compared with the tumor group;electroacupuncture improved post-chemotherapy bone marrow HSPCs（P<0.05）,LT-HSPCs（P<0.01）,MPPs（P<0.05）CLPs（P<0.05）,GMPs（P<0.001）,and MEPs（P<0.05）were reduced in number.Compared with the electroacupuncture group,the effect of electroacupuncture to increase the number of multiple stem progenitor cell populations in the bone marrow was lost after sympathetic nerve destruction,and the electroacupuncture+sympathetic nerve antagonism group showed a decrease in the number of HSPCs（P<0.01）,LT-HSPCs（P<0.001）,ST-HSCs（P<0.01）,MPPs（P<0.05）,CLPs（P<0.05）,GMPs（P<0.001）,and MEPs（P<0.05）were all reduced in number compared to the electroacupuncture group.Meanwhile,the number of bone marrow LT-HSPCs（P<0.001）,ST-HSCs（P<0.01）,GMPs（P<0.05）and the number of bone marrow cells in the G₀/G₁phase（P<0.05）were reduced in the mice in the electroacupuncture+PAC1antagonist group compared with the electroacupuncture group.The above results suggest that sympathetic and PACAP/PAC1 receptor pathways mediate electroacupuncture to improve the proliferative capacity of bone marrow hematopoietic cells in Lewis lung cancer chemotherapy mice.The results of nestin immunofluorescence staining and quantification of positive cell counts in the bone marrow of mice in each group showed that nestin⁺MSC expression decreased in the sympathetic nerve destruction compared with the electroacupuncture group（P<0.01）;nestin⁺MSC expression was similarly reduced in mice in the electroacupuncture+PAC1 antagonist group compared with the electroacupuncture group（P<0.001）.It was suggested that sympathetic and PAC1 receptors mediated the effect of electroacupuncture to increase the expression of nestin⁺mesenchymal stem cells in bone marrow of Lewis lung cancer chemotherapy mice,respectively.Conclusion1.Low-intensity electro-acupuncture at ST36 and SP6 can improve bone marrow suppression after chemotherapy in lung cancer mice,and can enhance the tumor inhibition effect of chemotherapy drugs.2.Sympathetic nerve and PACAP/PAC1 receptor pathways may be involved in electroacupuncture to improve the bone marrow hematopoietic microenvironment and hematopoietic function after chemotherapy in mice with lung cancer.