| Objective To investigate the protective effect of Pseudoprotodioscin?PPD?on bilateral oophorectomy?OVX?-induced cardiac function damage in apoE-/-mice.Methods?1?Eight weeks old,SPF,female,apoE knockout(apoE-/-)C57BL/6J mice were selected.The OVX advancement period was stable for one week?week 0?,and normal diet and water were given during the stable period.The mice were fed freely.In the first week,the mice were randomly divided into the sham operation group?Sham group?,the bilateral oophorectomy model group?OVX group?,the high dose PPD group?OVX+PPD1 group?after OVX,and the medium dose PPD group after OVX.?OVX+PPD2 group?,OVX group,low dose PPD group?OVX+PPD3 group?and OVX group,17?-estradiol?E2?group?OVX+E2 group?,8 mice in each group.The PPD was dissolved in DMSO to make a stock solution,which was stored at-20?with the refrigerator.Tribromoethanol?8 ml/kg?was placed in the supine position after anesthesia,disinfected with alcohol,1 cm at the left and right sides of the midline of the dorsal rib,1cm in the longitudinal incision,and the reddish-brown ovary surrounded by adipose tissue was found and ligated.After the uterine horn,bilateral oophorectomy was performed and the wound was sutured.The rest of the Sham group was the same as the OVX group except that the ovaries were not removed and the uterine horn was ligated.A high cholesterol diet?1.25%cholesterol?was then administered for 12 weeks.In the last 4 weeks,the next day was given:?1?Sham group:intraperitoneal injection?i.p.?saline containing 0.1%DMSO;?2?OVX group:intraperitoneal injection?i.p.?saline containing 0.1%DMSO;?3?OVX+PPD1 group:intraperitoneal injection?i.p.?4 mg/kg PPD;?4?OVX+PPD2 group:intraperitoneal injection?i.p.?2 mg/kg PPD;?5?OVX+PPD3 group:intraperitoneal injection?i.p.?1 mg/kg PPD;?6?OVX+E2 group:intramuscular injection?i.m.?0.5 mg/kg of E2.Dosing was stopped at week 13.?2?After the anesthesia of the mouse,the entire chest and abdomen were shaved with an electric hair clipper.To ensure the hair was removed,the shaving hair was applied twice with a depilatory cream.The chest and abdomen skin was wiped and disinfected by alcohol,fixed in High-Resolution In Vivo Imaging System,and the RMV7078 probe was selected for echocardiographic monitoring.The probe notch is oriented toward the head of the mouse,and rotated 30°to 45°counterclockwise to obtain the long-axis EKV of the parasternal side of the heart,which is stored in an animated form for tracing measurement.Detection of left ventricle internal diameter during diastole?LVIDd?,left ventricle internal diameter during systole?LVIDs?,internal ventricular septum during diastole?IVSd?,internal ventricular septum during systole?IVSs?,end-diastolic volume?EDV?,end-systolic volume?ESV?posterior wall thickness during diastole?PWTd?,posterior wall thickness during systole?PWTs?,ejection fraction?EF?,fractional shortening?FS?.?3?Combined with the results of TC and TG,2 mg/kg was used as the optimal dose of PPD for subsequent experiments.After anesthesia in mice,the right forelimb is connected to the negative electrode?-?,the left hind limb is connected to the positive electrode?+?,the biosignal acquisition system is turned on,and the electrocardiogram of the II lead is recorded by ECG.The detection time lasts for 30 min?intercept part?,and the PR interval is obtained.,QRS interval,R wave and heart rate data,reflecting the extent of heart damage after OVX.?4?Blood was taken from the heart of the mouse,plasma was prepared,and the absorbance value was measured at a wavelength of 510 nm.The plasma total cholesterol?TC?and triglyceride?TG?were measured according to the standard.?5?Cut the mouse chest to remove the heart,measure the wet weight of the heart,and take a photo of the heart.?6?Determination of plasma ultra-low-density lipoprotein cholesterol/intermediate density lipoprotein cholesterol?VLDL-C/IDL-C?,low-density lipoprotein cholesterol?LDL-C?,high-density lipoprotein cholesterol?HDL-C?concentration.The VLDL-C/IDL-C absorbance value was measured at a wavelength of 450 nm,and the absorbance values of LDL-C and HDL-C were measured at 546 nm.?7?Real-time quantitative RT-PCR was used to detect mRNA expression levels of atrial natriuretic factor?ANF?,tumor necrosis factor-??TNF-??,interleukin-1??IL-1??and interleukin-6?IL-6?.Total RNA was isolated using Trizol reagent and cDNA was synthesized using the PrimeScriptTM RT Master Mix kit.A real-time polymerase chain reaction is then performed.Analysis was performed using a real-time PCR analysis software,Applied Biosystems Primer Express Software?version 2.0?.Results?1?The blood lipid concentration results after 4 weeks of different doses of PPD and E2 showed that there was no significant difference in plasma TC concentration.TG content in the OVX group compared with the Sham group,increased by 45.57%?P<0.05?.After administration of different doses of PPD,the concentration of TC in OVX+PPD1 group was significantly lower than that in OVX group?P<0.05?,and the concentration of TG was not significantly decreased.The concentration of TC in OVX+PPD2 group was significantly lower than that in OVX group,which was decreased by 26.94%.?P<0.05?,the concentration of TG decreased significantly,decreased by69.12%?P<0.05?.There was no significant decrease in TC and TG concentrations in OVX+PPD3 group compared with OVX group.The effect of PPD?2 mg/kg?is similar to that of E2.?2?The hearts of the four groups of mice were weighed.Take a photo of the whole heart.The heart weight of the OVX group was significantly increased compared with the Sham group?P<0.05?.After PPD administration,the increase in heart weight of the mice was reversed compared with the OVX group?P<0.05?.?3?ECG test results showed that compared with Sham group,the heart rate of OVX group was decreased by 42.80%?P<0.05?,while PPD could inhibit the effect of OVX on heart rate,and heart rate increased by 75.94%?P<0.05?.The PR interval and QRS interval in the OVX group were increased by 14.52%and 47.62%,respectively?P<0.05?.PPD administration can inhibit the effects of OVX on both.The PR interval and QRS interval were decreased by 19.72%and 41.94%,respectively.?4?Echocardiographic data showed that LVIDd,LVIDs,IVSd,and IVSs in OVX group were increased by 6.67%,4.31%,5.19%,and 17.24%,respectively,compared with Sham group;OVX compared with OVX group The LVIDd and LVIDs of the OVX+PPD group were decreased by10.25%and 16.12%,respectively?P<0.05?,while the IVSd and IVSs were increased by9.88%and 5.88%,respectively.Compared with the Sham group,the EDV and ESV scores of the OVX group increased by 11.11%and 9.54%?P<0.05?.PPD inhibited the effect of OVX on the two groups,which was 19.99%and 31.53%lower than that of the OVX group?P<0.05?.For the PWTd,PWTs,EF,FS and other indicators,the OVX+PPD group increased by 15.87%,7.89%,6.09%,and 11.21%,respectively,compared with the OVX group.?5?Apolipoprotein content in mouse plasma showed no significant difference in plasma apolipoprotein?VLDL-C/IDL-C,LDL-C,HDL-C?concentrations in OVX group compared with Sham group.However,after administration of PPD,the concentration of VLDL-C/IDL-C in plasma was significantly lower than that of OVX group,which was decreased by 40.87%?P<0.05?,and the concentrations of LDL-C and HDL-C were decreased by 4.56%and 21.74.But there is no significant difference.?6?Real-time quantitative RT-PCR results showed that OVX significantly increased the mRNA levels of TNF-?,IL-1?,IL-6 and ANF in apoE-/-females compared with Sham group?P<0.05?.The increase in the expression of TNF-?,IL-1?,IL-6 and ANF was significantly reduced by 54.43%,51.29%,21.05%,and 34.98%,respectively,compared with the OVX group?P<0.05?.Conclusion?1?The use of 1,2,4 mg/kg of PPD,the OVX caused by the disorder of blood lipids have played a different degree of reversal,because the medium dose?2 mg/kg?PPD effect is the most significant,and E2 effect is the most Similarly,we determined that 2 mg/kg PPD was the optimal dose for the next study.?2?It can be seen from the data of echocardiography and electrocardiogram that the long-term high cholesterol diet after modeling leads to stress overload in the heart of the mouse,and the structure and function of the heart are abnormal,resulting in compensatory cardiac hypertrophy in mice.The heart rate and heart excitability disorder of the mice after PPD administration were improved,the time required for the atrium to start exciting to the ventricle and the time of change of the potential of the two ventricular excitatory processes were shortened,and the wall thickness and interventricular septum were significantly reduced.The myocardial contractility is enhanced,and the blood volume of the heart stroke is increased,which is similar to the administration of E2.This proves that PPD can effectively alleviate the damage of cardiac function induced by cholesterol diet after OVX.?3?The effect of mouse OVX on VLDL-C/IDL-C,LDL-C,HDL-C is not significantly different,although both PPD and E2 can significantly down-regulate VLDL-C/IDL-C,the protective effect of plasma apolipoprotein on the heart is not clear.Therefore,the alleviation of PPD on heart damage may not be explained by plasma apolipoprotein.?4?The mRNA expression levels of inflammatory factors TNF-?,IL-6 and IL-1?and cardiomyocyte hypertrophic marker ANF were abnormally increased after OVX,while PPD could effectively reduce the mRNA expression level,similar to E2.Consider PPD to reverse the cardiac damage of OVX through anti-inflammatory effects.In conclusion,PPD has a protective effect on OVX-induced cardiac function damage in apoE-/-mice. |
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