ABSTRACT
OBJECTIVE：To study the effects of Calpeptin inhibitor Calpeptin on the transformation and stemness markers expression induced by estradiol（E2），and to investigate its mechanism. METHODS ：Taking human mammary epithelial cells MCF-10A as research object ，transformed cells were induced by E 2 treatment. Cells were divided into control group （0.1％DMSO）， E2-transformed group （50 nmol/L），E2-transformed+Calpeptin group （50 nmol/L E 2+1 μmol/L Calpeptin），then continuously treated with corresponding drug-containing culture medium for 15 generations. Then ，MTT assay was used to determine the proliferation rate of cells （24，48 h）；plate colony test was used to detect the Clone formation rate of cells ；the number of sphere-forming cells was measured by suspension spheroidization test ；mRNA expressions of stemness marker （CD44，Nanog，OCT4）and extracellular sigal-regulated kinase （ERK）were detected by RT-qPCR ，and protein expressions of CD 44，Nanog，OCT4 ，ERK and p-ERK were detected by Western blotting assay. Another E 2-transformed cells were divided into control group （0.1％DMSO）and U0126 （ERK inhibitor ）group（10 μmol/L）. Clone formation rate ，the number of sphere-forming ，protein expressions of CD 44，Nanog， OCT4，ERK and p-ERK were determined with above methods ，and to validate the relationship of ERK inhibition with transformed cell behavior and the expression of stemness markers. RESULTS ：Compared with control group ，proliferation rate and clone formation rate of E 2 transformed group were increased significantly （P＜0.01），and the number of sphere-forming was increased significantly（P＜0.01）；mRNA expression levels of CD 44，Nanog，OCT4，ERK and protein expression levels of CD 44，Nanog， OCT4 and p-ERK in cells were increased significantly （P＜0.01）. Compared with E 2-transformed group ，proliferation rate （24，48 h）and clone formation rate of E 2-transformed + Calpeptin group were decreased significantly （P＜0.01），and the number of sphere-forming was decreased significantly （P＜0.05）；mRNA expression levels of CD 44，Nanog，OCT4 ，ERK and protein expression levels of CD 44，Nanog，OCT4，p-ERK in cells were decreased significantly （P＜0.05 or P＜0.01）. After treated with ERK inhibitor U 0126，clone formation rate of E 2-transformed cells ，the number of sphere-forming ，protein expression levels of CD44，Nanog，OCT4 and p-ERK were increased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：Calpeptin can inhibit the transformation and the expression of stemness markers of human mammary epithelial cells MCF- 10A，and the mechanism of it may be associated with inhibiting the activation of Calpain-ERK signaling pathway.
ABSTRACT
Aim To explore the effects of acupuncture and sinomenine on the withdrawal symptoms,on the spontaneous discharge and synaptophysin expression of amygdala of heroin-addicted rats.Methods 75 SD rats were randomly and equally divided into 5 groups consisted of control,heroin-addicted,acupuncture,sinomenine and combined treatment group.The model of heroin-addicted rats were established with heroin subcutaneous injection,which in acupuncture,sinomenine and combined group were subsequently punctured at 3 acupoint(Neiguan,Baihui and Zusanli),intramuscular injected with sinomenine and treated with combined methods(acupuncture and sinomenine)respectively.The withdrawal symptoms of rats in different group were evaluated,and then the spontaneous discharges and synaptophysin expression of amygdala were detected with electrophysiological and immunohistochemical technology respectively.Results The heroin-addicted rats were successfully established,being suggested by typical withdrawal symptoms induced by naloxone(P<0.01 vs control),which were alleviated in the 3 treatment groups(acupuncture,sinomenine and combined ) with varying degree(P<0.01 vs heroin group).The electrophysiological results suggest that the forms of low-frequency and single irregular discharges in amygdala were increase,bunchy/cluster and complex discharges were decreased(P<0.05 vs control),which were reversed apparently in sinomenine and combined treatment groups(P<0.05 vs heroin group),closed to control.The immunohistochemical results show that expression of synaptophysin in amygdala was increased obviously(P<0.01 vs control),which was decreased in the 3 treatment groups(acupuncture,sinomenine and combined),P<0.01 vs addicted group.Conclusion Treatments of acupuncture and sinomenine administration can alleviate withdrawal symptoms of heroin-addicted,and rectify the abnormal spontaneous discharges and expression of synaptophysin in amygdala of heroin-addicted rats for certain degree,especially with combined treatments.