Iron Overload Promotes Myocardial Fibrosis In Mice With Renal Failure Via MLK3 Signaling

Objective:To determine whether iron overload can promote myocardial fibrosis in renal failure mice via MLK3。Methods:1.In-vivo experiments:C57 mice were divided into 5 groups with 15 mice in each group:sham-operated group（Sham）,renal failure operated group（CRF）,iron overload group（CRF+Fe）,MLK3 inhibited group（CRF+Fe+URMC-099）,and MLK3 agonisted group(CRF+Fe+URMC-099+AAV^MLK3-).In this study,a mouse model of renal failure was constructed using the 5/6 nephrectomy method,and the levels of serum creatinine and urea nitrogen in the sham-operated and operated groups were measured 12 weeks after surgery to assess the presence or absence of renal failure in both groups,as well as the presence or absence of renal injury in both groups by HE staining of kidney tissues.After successful preparation of the sham-operated,renal failure-operated,iron-overloaded,MLK3-inhibited and MLK3-excited groups,the blood and tissue specimens of each group were collected to assess the degree of oxidative stress in the serum.After the blood and tissue specimens were collected from each group,the level of oxidative stress in the serum was assessed by measuring the level of MDA and SOD in the serum.The fluorescence probe DHE was used to assess the level of oxidative stress in myocardial tissue,and tissue immunofluorescence and Western Blot were used to determine the expression ofα-smooth muscle actin（α-SMA）,vimentin and other indicators of myocardial fibrosis and their correlation with MLK3 signalling proteins.2.In vitro experiments:Cell experiments were divided into four groups:control group（Sham）,iron overload group（Fe）,MLK3 inhibition group（Fe+URMC-099）,and MLK3activation group（Fe+URMC-099+rm MLK3）.After adding iron dextran,URMC-099,and exogenous rm MLK3 stimulation respectively,it was used to construct an iron overload group,an MLK3 inhibition group,and an MLK3 activation group.Western blot was used to detect the expression of related proteins,and immunofluorescence was used to detect the myocardial fibrosis related proteins in the myocardium of mice in each groupα-Smooth muscle actin(α-Expression of SMA and vimentin was observed to observe the transdifferentiation of cardiac fibroblasts,and the effect of MLK3 on the migration ability of cardiac fibroblasts was evaluated by cell scratch test.Fluorescent probe DCFH-DA was used to detect the level of ROS oxidative stress in mouse cardiac fibroblasts.Results:1.The serum creatinine and serum urea nitrogen of the mice in the renal failure group and the sham-operated group were measured 12 weeks after surgery,and It can be found that the serum creatinine and serum urea nitrogen in renal failure operated group significantly increase（P<0.01）.In comparison with the sham-operated group,the renal tubules of the mice in the renal failure group were clearly dilated and atrophied,with different cavities in the tubular lumen and an increase in fibroblasts and collagen fibres,as seen on the HE staining of the renal tissues of the sham-operated and renal failure groups.2.Prussian blue staining of heart sections showed that iron deposition was evident in the myocardial tissue of the mice in the iron overload group compared to the renal failure and sham-operated groups,while iron deposition was not evident or even absent in the cardiac tissue of the renal failure and sham-operated groups.HE staining of mouse heart tissues showed that the myocardium of the sham-operated group was neatly and evenly arranged,while the myocardium of the operated group was slightly less neatly arranged,and the myocardium of the iron-overloaded group was clearly disordered and fractured.3.HE staining showed that compared with the intact myocardial structure in the sham operated group,the myocardial tissue arrangement in the renal failure group was slightly irregular,while after iron supplementation,the myocardial tissue arrangement in the mice was disorderly,and even some muscle filaments were broken.4.Masson staining experiments showed that only a small amount of blue collagen fibres were deposited between cells in the renal failure group compared to the sham-operated group,whereas a large amount of blue collagen fibres were seen between heart tissue in the iron overload group established after 12 weeks of iron supplementation in the renal failure group.The blue collagen fibres were significantly reduced in the MLK3 inhibited group compared to the iron overload group.In contrast,the proportion of collagen fibres was significantly increased in the MLK3 agonist group compared to the MLK3 inhibited group.5.Western Blot showed that the expression of MLK3（P<0.001）and myocardial fibrosis-associated proteinα-SMA（P<0.01）was significantly higher in the renal failure group compared with the sham-operated group,while vimentin was not significantly higher in the renal failure group compared with the sham-operated group.The iron overload group had significantly higherα-SMA（P<0.01）,vimentin（P<0.05）and MLK3（P<0.01）proteins compared to the renal failure group,and the difference was statistically significant（P<0.05）.The expression of MLK3 andα-SMA and vimentin,which are indicators of myocardial fibrosis,were significantly reduced in the MLK3 inhibited group compared to the iron overload group（P<0.05）.The expression of MLK3（P<0.01）and myocardial fibrosis-related indicator proteinsα-SMA（P<0.01）and vimentin（P<0.001）was increased in the MLK3 agonist group compared to the MLK3 inhibitor group（P<0.05）.The results of the quantitative analysis of the fibrosis marker proteinsα-SMA and vimentin by immunofluorescence staining were in general agreement with the Western Blot results.6.ROS staining was used to detect the expression of ROS,MDA,and SOD in cardiac tissue of mice in each group to observe the level of reactive oxygen species in myocardial cells.The results showed that there was no significant difference in red fluorescence between the renal failure group and the sham operation group.Compared with mice in the renal failure group,myocardial tissue in the iron overload group showed strong red fluorescence（P<0.05）,while ROS production was reduced in the MLK3 inhibition group（P<0.05）.After tail vein injection of AAVMLK3-,the expression of ROS in the MLK3 agonist group increased significantly（P<0.05）,and the trend of MDA and SOD expression was basically consistent with the results of ROS expression.7.In vitro culture of cardiac fibroblasts with different concentrations of iron dextran,Western Blot results showed that when the concentration of iron dextran reached 100μmol/L,α-The expression levels of SMA,vimentin,and MLK3 proteins were higher than the concentrations of 0,10,50,and 200μmol/L,reaching the highest level.Therefore,iron dextran with a concentration of 100μmol/L was selected as the optimal iron concentration for this experiment.Using the same method and procedure,the optimal action concentration for determining rm MLK3 is 50μmol/L.8.Western Blot results ofα-SMA and vimentin,the central myofibrosis-related index proteins,in the control group,iron overload group,MLK3 inhibition group and MLK3agitation group showed that the expression ofα-SMA（P<0.01）,vimentin（P<0.01）and MLK3（P<0.001）was significantly reduced in the MLK3 inhibition group compared to the iron overload group,while the expression of vimentin and MLK3 was significantly increased in the MLK3 agitation group compared to the MLK3 inhibition group（P<0.01）.The expression of vimentin and MLK3 was significantly higher in the MLK3 agonist group compared to the MLK3 inhibitor group（P<0.01）.9.The results of the scratch assay showed that the scratch healing rate of myocardial fibroblasts was significantly lower in the MLK3 inhibited group compared to the iron overloaded group（P<0.01）,while the scratch healing rate was significantly higher in the MLK3 agonist group compared to the MLK3 inhibited group（P<0.05）.10.When ROS expression was measured in cells cultured in vitro,myocardial tissue showed a strong red fluorescence（P<0.05）in the iron-loaded group compared to the control group,while ROS expression was significantly reduced（P<0.05）in the MLK3-inhibited group and significantly increased（P<0.05）after the reintroduction of exogenous rm MLK3.Conclusions:1.Iron overload promotes myocardial fibrosis in mice with renal failure by agonizing MLK3 signaling.2.Iron overload promotes migration and conversion of cardiac fibroblasts to myofibroblasts by agonizing MLK3 signaling.