The Effect Of Doxycycline On TGF-β1,MMP-2 And TIMP-1 Expression In Rats With Arterial Calcification

Background and Objective:Arterial calcification means the deposition of calcium phosphate in the form of apatite in the media of arteries.AC is thought to be a key contributor to cardiovascular morbidity and mortality,it leads to increase in arterial stiffness and decrease in arterial compliance,resulting in hypertension,aortic stenosis,left ventricular hypertrophy,congestive heart failure,and possibly plaque rupture,thrombosis,and myocardial infarction.However,the precise mechanism of arterial calcification has not yet been elucidated.Thus,in the current study,we showed induction of AC in rats following the application of excessive vitamin D₃subcutaneous injection,We set doxycycline as a drug intervention and observed the effect of doxycycline on the formation of arterial calcification and the expression of TGF-β1,MMP-2 and TIMP-1,with the expectation to find a new prophylaxis and therapy clue to the formation of arterial calcification.Materials and Methods:Sixty seven-week-old SPF male Sprague-Dawley rats were randomly divided into four groups（n=15 for each group）:control group（A）,model group（B）,doxycycline 60 mg/kg intervention group（C）and doxycycline 120 mg/kg intervention group（D）.The rat calcification models were established by subcutaneous injection of vitamin D₃ 600000 U·kg-1 once a day for 3 days.The rat calcification models were established as above in drug intervention group and at the same time the rats were provided with 60,120 mg/kg doxycycline daily by subcutaneous injection beginning at 2 days prior to vitamin D₃injections and ending at the time of harvest.The control group and model group underwent the same procedure,except that animals received subcutaneous injection of normal saline.All rats were killed two weeks after the first doxycycline injection,and the aorta from the arch to the iliac bifurcation was harvested.Some fresh aorta tissues were fixed in formalin solution for pathological analysis,and others were maintained immediately in liquid nitrogen for further evaluation.The change of arterial tissue in different groups was decided by Hematoxylin and eosin（HE）staining,arterial calcification was assessed by Von Kossa staining and calcium content was determined by colorimetry through a reaction with o-cresolphthalein complexon.The expression level of TGF-β1,MMP-2 and TIMP-1 m RNA and protein were detected by RT-q PCR and western blot,respectively.Results:1.From HE staining we can see normal structure of artery tissues,and the smooth muscle cells in tunica media were arranged in linear in the control group,whereas the model group contains a disordered arrangemen of vascular smooth muscle cells and fracture of elastic fibres;The intervention group has smaller scale of artery injury,the damage of medial artery elastic fiber is not apparent.2.Strong positive Von Kossa staining for calcium deposits among the elastic fibers of the medial layer was found in the aorta of model group,However,Black granules corresponding to calcium phosphate deposits were obviously decreased in rats treated with doxycycline in a dose-dependent manner compared to model rats.No obvious positive Von Kossa staining was found in a negative control section.3.Compared with the control group,the aortic calcium contents in the model group was significantly increased.Doxycycline treatment decreased the aortic calcium contents compared with the model group.Moreover,doxycycline treatment with 120 mg/kg was more effective in decreasing aortic calcium contents than treatment with 60 mg/kg.4.Real-time q PCR revealed increased m RNA expression of TGF-β1,MMP-2 and TIMP-1 m RNA level in model group,as compared with control group.However,doxycycline treatment can significantly reduce the TGF-β1 and MMP-2 m RNA expression but increased the TIMP-1m RNA level.5.Western blot showed that TGF-β1,MMP-2 and TIMP-1 protein expressions were significantly increased in model group compared with control group,doxycycline treatment significantly reduced the increased TGF-β1 and MMP-2 protein levels.In contrast,the TIMP-1 protein expressions were increased after doxycycline treatment.Conclusions:Taken together,these data demonstrate that TGF-β1,MMP-2 and TIMP-1 play a pivotal role in the pathological process of artery calcification.As artery calcification advances,TGF-β1,MMP-2and TIMP-1 gradually enhance,which reflects the obvious enhancement of the degradation and synthesis of extracellular matrix.Doxycycline can effectively attenuated artery calcification by inhibiting the expression of TGF-β1 and MMP-2 but promoting the expression of TIMP-1.