Mibefradil improves skeletal muscle mass, function and structure in obese mice / 南方医科大学学报

OBJECTIVE@#To observe the effect of mibefradil on skeletal muscle mass, function and structure in obese mice.@*METHODS@#Fifteen 6-week-old C57BL/6 mice were randomized equally into normal diet group (control group), high-fat diet (HFD) group and high-fat diet +mibefradil intervention group (HFD +Mibe group). The grip strength of the mice was measured using an electronic grip strength meter, and the muscle content of the hindlimb was analyzed by X-ray absorptiometry (DXA). Triglyceride (TG) and total cholesterol (TC) levels of the mice were measured with GPO-PAP method. The cross-sectional area of the muscle fibers was observed with HE staining. The changes in the level of autophagy in the muscles were detected by Western blotting and immunofluorescence assay, and the activation of the Akt/mTOR signaling pathway was detected with Western blotting.@*RESULTS@#Compared with those in the control group, the mice in HFD group had a significantly greater body weight, lower relative grip strength, smaller average cross sectional area of the muscle fibers, and a lower hindlimb muscle ratio (P < 0.05). Immunofluorescence assay revealed a homogenous distribution of LC3 emitting light red fluorescence in the cytoplasm in the muscle cells in HFD group and HFD+Mibe group, while bright spots of red fluorescence were detected in HFD group. In HFD group, the muscular tissues of the mice showed an increased expression level of LC3 II protein with lowered expressions of p62 protein and phosphorylated AKT and mTOR (P < 0.05). Mibefradil treatment significantly reduced body weight of the mice, lowered the expression level of p62 protein, and increased forelimb grip strength, hindlimb muscle ratio, cross-sectional area of the muscle fibers, and the expression levels of LC3 II protein and phosphorylated AKT and mTOR (P < 0.05).@*CONCLUSION@#Mibefradil treatment can moderate high-fat diet-induced weight gain and improve muscle mass and function in obese mice possibly by activating AKT/mTOR signal pathway to improve lipid metabolism and inhibit obesityinduced autophagy.

Effect of mibefradil on proliferation of human pulmonary artery smooth muscle cells induced by platelet-derived growth factor / 临床儿科杂志

Objectives To explore the effect of mibefradil, a kind of novel calcium channel antagonists, on proliferation of human pulmonary artery smooth muscle cells (HPASMCs) induced by platelet-derived growth factor (PDGF). MethodsHPASMCs were culturedin vitro, and randomly divided into control group, PDGF group, Mib group, and PDGF+Mib group. The PDGF group was stimulated by 25 ng/ml of PDGF. Mib group was intervented by 10 μmol/L of Mib. PDGF+Mib group was treated by PDGF and Mib. The reproduction rate in 48 hours and 72 hours were detected by MMT. Cell cycle was detected by lfow cytometry. The expression of proliferating cell nuclear antigen (PCNA) was observed by immunolfuorescence staining (IFS).ResultsThere were statistical differences among four groups in both 48 hours and 72 hours (P all??0.05). There were statistically differences of G0/G1 phase and S phase among four groups (P?

Impact of calcium channel antagonists for estrogen action on the endometrial carcinoma HEC-1A cells / 中华妇产科杂志

Objective To study the effects of nifedipine and mibefradil on the cell proliferation,apoptosis,migration on the HEC-1 A in vitro and also study the mRNA and protein expression levels of the calcium channel alpha1 D( Cav1.3 ) and calcium channel alpha1G (Cav3.1 ) to discuss the effects of the calcium antagonists on the mechanisms of the endometrial carcinoma.Methods ( 1 ) Add 10 μmol/L nifedipine and mibefradil at 15 minutes before adding 10 μmol/L 17β-estradiol( 17β-E2 ) and 100 μmol/L b-estradiol-6-(O-carboxymethyl) oxime(E2-BSA) to the HEC-1A in different time including 0,5,15,30,60,120 minutes.Then the changes of mRNA and protein were detected by reverse transcripiton( RT)-PCR and western-blot.(2)Add 1.25,2.5,5,10,20,40,80,100 μmol/L nifedipine and mibefradil to the HEC-1 A at 24,48,96 hours to detect the cell proliferation by 3-( 4,5 )-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide (MTT) method.(3) Add 10 μmol/L nifedipine and mibefradil to the HEC-1A,then detect the apoptosis at 0 minute,30 minutes,1 hour,6 hours,24 hours and migration in vitro at 36 hours with transwell methods.Results ( 1 ) After the pretreated effect of the nifedipine before 17β-E2,the mRNA express of Cav1.3 genes was lowest at 15 minutes,and returned to the control level after 30 minutes.The protein level didn't change very much in 30 minutes,but rose after 60 minutes.The Cav3.1 genes mRNA express was lowest at 5 minutes,rose at 30 minutes and returned to the 0 minute level gradually.(2) After the pretreated effect of the nifedipine before E2-BSA,the Cav1.3 genes mRNA was lowest at 5 minutes and returned at 15 minutes.The protein level rose gradually in 15 minutes but reduced after 15 minutes.The Cav3.1 mRNA and protein level were reduced at every time point.(3) After the pretreated effect of the mibefradil before 17β-E2,there was no change of mRNA expression of Cav1.3 genes.The protein level rose at 15 and 60 minutes,there was no change in any other time.The Cav3.1 genes mRNA were gradually reduced and the protein level rose at 15 minutes,and there was no change in any other times.(4) After the pretreated effect of the mibefradil before E2-BSA,the mRNA and protein of Cav1.3 levels were reduced after 15 minutes.There was no mRNA expression of Cav3.1,while the protein level was lowest at 15 minutes.(5)Nifedipine and mibefradil affected HEC-1A proliferation depended on the different concentration and interval time points.There was significant difference than those in control group ( P ＜ 0.05 ).( 6 ) There were statistical differences in apoptosis rate after adding nifedipine ( P ＜ 0.05 ),while rose at mibefradil treated the same time ( 24 hours:8.41 ± 0.07,0 minute:3.74 ± 0.18 ; P ＜ 0.05 ).(7) The numbers of stained cells after both nifedipine and mibefradil treated reduced more than control group.Conclusions ( 1 ) Nifedipine and mibefradil could inhibit both the effect of the estrogen on the L-type and T-type calcium channel in short time,meanwhile the mibefradil effects last long time. (2) The inhibited effect of the mibefradil on the proliferation,apoptosis and migration of HEC-1A cells in vitro is more significant than that by nifedidipine.

Role of T-type Ca2+ Channels in the Spontaneous Phasic Contraction of Pregnant Rat Uterine Smooth Muscle

Although extracellular Ca2+ entry through the voltage-dependent Ca2+ channels plays an important role in the spontaneous phasic contractions of the pregnant rat myometrium, the role of the T-type Ca2+ channels has yet to be fully identified. The aim of this study was to investigate the role of the T-type Ca2+ channel in the spontaneous phasic contractions of the rat myometrium. Spontaneous phasic contractions and [Ca2+]i were measured simultaneously in the longitudinal strips of female Sprague-Dawley rats late in their pregnancy (on day 18~20 of gestation: term=22 days). The expression of T-type Ca2+ channel mRNAs or protein levels was measured. Cumulative addition of low concentrations (<1 micrometer) of nifedipine, a L-type Ca2+ channel blocker, produced a decrease in the amplitude of the spontaneous Ca2+ transients and contractions with no significant change in frequency. The mRNAs and proteins encoding two subunits (alpha1G, alpha1H) of the T-type Ca2+ channels were expressed in longitudinal muscle layer of rat myometrium. Cumulative addition of mibefradil, NNC 55-0396 or nickel induced a concentration-dependent inhibition of the amplitude and frequency of the spontaneous Ca2+ transients and contractions. Mibefradil, NNC 55-0396 or nickel also attenuated the slope of rising phase of spontaneous Ca2+ transients consistent with the reduction of the frequency. It is concluded that T-type Ca2+ channels are expressed in the pregnant rat myometrium and may play a key role for the regulation of the frequency of spontaneous phasic contractions.

Antihyperalgesic Effects of Ethosuximide and Mibefradil, T-type Voltage Activated Calcium Channel Blockers, in a Rat Model of Postoperative Pain / 대한통증학회지

BACKGROUND: A correlation between a T-type voltage activated calcium channel (VACC) and pain mechanism has not yet been established. The purpose of this study is to find out the effect of ethosuximide and mibefradil, representative selective T-type VACC blockers on postoperative pain using an incisional pain model of rats. METHODS: After performing a plantar incision, rats were stabilized on plastic mesh for 2 hours. Then, the rats were injected with ethosuximide or mibefradil, intraperitoneally and intrathecally. The level of withdrawal threshold to the von Frey filament near the incision site was determined and the dose response curves were obtained. RESULTS: After an intraperitoneal ethosuximide or mibefradil injection, the dose-response curve showed a dose-dependent increase of the threshold in a withdrawal reaction. After an intrathecal injection of ethosuximide, the threshold of a withdrawal reaction to mechanical stimulation increased and the increase was dose-dependent. After an intrathecal injection of mibefradil, no change occurred in either the threshold of a withdrawal reaction to mechanical stimulation or a dose-response curve. CONCLUSIONS: The T-type VACC blockers in a rat model of postoperative pain showed the antihyperalgesic effect. This effect might be due to blockade of T-type VACC, which was distributed in the peripheral nociceptors or at the supraspinal level. Further studies of the effect of T-type VACC on a pain transmission mechanism at the spinal cord level would be needed.