艾灸足三里、关元通过外泌体递送微小RNA-298-5p调控乳腺癌增殖的实验研究OA

Objective:Breast cancer is the most common malignancy among women worldwide.Moxibustion,as an external treatment in traditional Chinese medicine,has shown potential in adjuvant cancer therapy.This study aims to elucidate the mechanism by which moxibustion at Zusanli(ST36)and Guanyuan(CV4)regulates breast cancer proliferation through exosome-mediated delivery of target microRNAs,thereby providing experimental evidence for the application of moxibustion in breast cancer prevention and treatment.Methods:The study consisted of two parts.Part Ⅰ:A total of 36 female 12-week-old MMTV-PyMT spontaneous breast cancer mice were randomly divided into three groups:model group,sham-moxibustion group,and moxibustion group.Mice in the model group were raised under standard conditions;those in the sham-moxibustion group received only isoflurane inhalation anesthesia and fixation;and those in the moxibustion group received mild moxibustion at Zusanli and Guanyuan on the basis of the sham-moxibustion procedure.The intervention lasted for 42 consecutive days.Tumor volumes were measured at 12,15,and 18 weeks of age.Subsequently,serum samples were collected from the moxibustion and sham-moxibustion groups,and exosomes were isolated for microRNA sequencing.Part Ⅱ:Nine 12-week-old mice of the same strain were randomly divided into three groups:sham-moxibustion exosome group(Sham-Exo),moxibustion exosome group(Mox-Exo),and moxibustion exosome plus inhibitor group(Mox-Exo+Inh).Mice received tail vein injections of the corresponding exosomes or inhibitor-loaded moxibustion exosomes once weekly for four weeks.To clarify the mediating role of exosome-derived miR-298-5p in the anti-tumor effect of moxibustion,real time quantitutive poly-merase chain reaction(RT-qPCR)was performed to assess the intervention efficacy of the miR-298-5p inhibitor plas-mid.On day 48,mice were euthanized,and tumor tissues were collected and weighed to compare tumor weights a-mong the groups.Results:Results from Part Ⅰ showed that at 12 weeks of age,there were no significant differences in tumor volume among the three groups(all P＞0.05),indicating good comparability.At 15 and 18 weeks of age,tumor volumes in the moxibustion group were significantly smaller than those in the model group and the sham-mox-ibustion group(all P＜0.01),whereas no significant differences were observed between the model group and the sham-moxibustion group(P＞0.05),suggesting that anesthesia and fixation procedures themselves did not affect tumor growth.MicroRNA sequencing of exosomes revealed that miR-298-5p exhibited the most significant change in expression and was selected as the core target.Results from Part Ⅱ showed that RT-qPCR analysis demonstrated a significant downregulation of miR-298-5p expression in serum exosomes of the Mox-Exo+Inh group compared with the Mox-Exo group(P＜0.001),while no significant difference was observed compared with the Sham-Exo group(P＞0.05),confirming that the inhibitor plasmid effectively antagonized the high expression of miR-298-5p in moxi-bustion-derived exosomes.On day 42,tumor weight in the Mox-Exo group(0.46±0.08)g was significantly lower than that in the Sham-Exo group[(3.47±0.74)g,P＜0.001].The average tumor weight in the Mox-Exo+Inh group was(1.94±0.08)g,which was significantly higher than that in the Mox-Exo group(P＜0.05)but remained significantly lower than that in the Sham-Exo group(P＜0.05).Conclusion:Moxibustion at Zusanli and Guanyuan effectively inhibits breast cancer growth in MMTV-PyMT mice,and its mechanism of action may be related to the upregulation of miR-298-5p expression in circulating exosomes.Inhibition of miR-298-5p attenuates the tumor-sup-pressive effect of moxibustion-derived exosomes,suggesting that the"moxibustion-exosome-miR-298-5p"regulatory axis plays an important role in mediating the anti-tumor effect of moxibustion.