Advanced Glycation End Products Induce Apoptosis And Its Possible Mechanisms

BackgroundGastrointestinal(GI)motility disorder is one of the most common complications in diabetic patients.The pathogenesis of GI dysmotility in diabetes is still unclear and it is related with multiple factors,such as autonomic neuropathy,decreased number of interstitial cells of Cajal(ICCs),and lesions of smooth muscle cells(SMCs).Advanced glycation end products(AGE)accumulate in diabetic patients due to excess availability of simple 3-or 4-carbon sugars.AGE has multiple deleterious effects including autophagy,apoptosis,inflammation and obesity.It is important to understand the mechanism of AGE increased cell apoptosis,one such deleterious effect.In this study,we investigated the effects of receptor of AGEs(RAGE)and JNK,p38 MAPK on AGE-modified bovine serum albumin(BSA)induced colonic smooth muscle cells of SD rats apoptosis.Objective1.To investigate the expression of AGE and Apoptosis in diabetic colonic muscle tissues.2.To investigate the effect of AGEs/RAGE on MAPK pathway in colonic SMCs Apoptosis and its possible mechanisms.Methods1?Tissues preparation:1)Detection of AGE levels in serum and colonic muscle tissues of rats:Serum CML content was detected by centrifugal upper peripheral venous blood of rats;CML content in rats colon smooth muscle layer was detected by immunohistochemical.2)Detection of apoptosis in rat colon smooth muscle layer and proteins expression:Rat colonic tissue was made into wax block specimens.Apoptosis was measured by TUNEL kits.Smooth muscle layer was removed from the serosa and mucosa and then was cut into pieces.The protein expression levels of apoptosis-related genes were measured by western blot.2?Isolation and culture of colonic SMCs:The colon was taken from male SD rats.Smooth muscle layer was removed from the serosa and mucosa and then was cut into pieces.SMCs were isolated by enzymatic digestion and cultured with DMEM.1)Effect of AGEs on apoptosis,Caspase 3 activity and RAGE protein expression:Colonic SMCs were cultured with different concentrations of AGEs.Apoptosis was detected by apoptosis kit.Caspase 3 activity was tested by Caspase 3 activity assay kit.Western blot were used to examine the protein expression levels of RAGE.2)Effect of AGEs on apoptosis related proteins:Colonic SMCs were cultured with different concentrations of AGEs.Western blot were used to detect the expression of apoptosis related proteins.3)Effect of AGEs on MAPK signal pathway in SMCs:Colonic SMCs were cultured with optimal concentration of AGEs for different times,the protein expression levels in MAPK pathway were measured by western blot.4)Effect of RAGE receptor on MAPK signal pathway mediated,AGE-induced apoptosis in SMCs:Colonic SMCs were treated with siRNA(control siRNA and RAGE siRNA)for transfection before exposure to AGEs.Western blot were used to examine the protein expression levels in MAPK pathway.Results1.The expression level of AGE in colonic muscle tissues and serum:Compared with the control group,the expression of CML was significant increased in diabetic colonic muscle tissues especially mucosal muscularis and muscle layer.CML content of peripheral venous blood in DM group rats was significantly higher than the control as well.2.Apoptosis of rat colonic SMCs:Compared with control group,the apoptosis of colonic SMCs in DM group was significantly higher[(3.00±0.82)vs.(18.60±1.70),P<0.05].3.Expression level of Caspase 3,Bcl-2 and Bax in colonic tissue:Compared with control group,the expression of Caspase 3,Bax/Bcl-2 ratio in diabetic colonic muscle tissues was significant increased[(2.73±0.10)vs.(0.09+0.11)],[(0.15±0.02)vs.(1.24±0.12),P<0.05].4.The effect of AGEs on SMCs apoptosis:AGEs at the concentration of 100 ?g/mL,150 ?g/mL or 300 ?g/mL significantly increased SMCs apoptosis compared with the control group[(5.395±0.40)vs.(8.88±1.74)?(9.87±2.14)?(20.15±6.29),P<0.05],while there was no significance at the concentration of 50?g/mL(P>0.05).5.The effect of AGEs on Caspase 3 activity:AGEs at the concentration of 50 u g/mL,100 ?g/mL,150 ?g/mL or 300 ?g/mL significantly increased Caspase 3 activity compared with the control group[(0.45±0.07)vs.(0.62±0.03)?(0.71±0.03)?(0.80±0.03)?(1.01±0.05),P<0.05],while there was no significance at the concentration of 0?g/mL(P>0.05).6.The effect of AGE with different concentrations and time on Caspase 3 expression level:AGEs at the concentration of 50?g/mL,100?g/mL,150?g/mL or 300?g/mL significantly increased Caspase 3 protein expression compared with the control group[(0.17±0.05)vs.(0.17±0.06)?(0.29±0.08)?(0.33±0.11)?(0.35±0.10)?(0.36±0.10),P<0.05],while there was no significance at the concentration of 0?g/mL(P>0.05).AGEs at the time of 24h,36h,72h significantly increased Caspase 3 expression compared with the control group[(0.54±0.07)vs.(0.99±0.10)?(0.92±0.08)?(0.87±0.09)],P<0.05].7.The effect of AGE with different concentrations on expression of Bcl-2,Bax and RAGE:AGEs at the concentration of 100?g/mL,150?g/mL or 300?g/mL significantly increased RAGE expression compared with the control group[(0.56±0.08)vs.(0.87±0.09),(0.94±0.13),(1.00±0.10),P<0.05],AGEs at the concentration of 50?g/mL,100?g/mL,150?g/mL or 300?g/mL significantly increased Bax/Bcl-2 expression compared with the control group[(0.35±0.02)vs.(0.58±0.03)?(0.53±0.07)?(0.77±0.06)?(1.42±0.11),P<0.05],while there was no significance at the concentration of 0?g/mL(P>0.05).8.The effect of AGEs with different time on MAPK pathway:Compared with the control group,AGEs with 30min and 60min can increase the protein expression of p-P38 and p-JNK[(0.77±0.09)vs.(1.31±0.14)?(2.47±0.30),P<0.05],[(0.29±0.10)vs.(0.74±0.17),P<0.05].9.The effect of MAPK pathway in RAGE mediated apoptosis of colonic SMCs:Compared with control siRNA-AGE(300?g/mL,60 min)group,RAGE siRNA-AGE significantly decreased p-P38 expression[(1.21±0.18)vs.(0.35±0.09),P<0.05]and p-JNK expression[(1.06±0.12)vs.(0.42±0.23),P<0.05].The expression of p-ERK was no statistically significant difference between the two groups(P>0.05).RAGE siRNA inhibited the expression of P38 and JNK protein phosphorylation level.Conclusions1?Expression of AGE in diabetic colonic muscle tissues and serum is increased,as well as the apoptosis.2?AGE/RAGE induces apoptosis of colonic SMCs,and the mechanism involves the activity of MAPK pathway.