Therapeutic Effect And Mechanism Of Caffeine On Diabetic Cystopathy In Rats

Background and objective:The incidence of diabetes mellitus(DM)is increasing,and its complication,diabetic cystopathy(DCP)seriously affects the quality of life of patients.It is still a difficult point in its research field.The main mechanism of DCP is the myogenic and neurogenic lesions caused by chronic hyperglycemia lead to impaired contractile function of the detrusor.Caffeine is a methylxanthin,acts as an agonist of the sarcoplasmic reticulum Ry R receptor in the cell,regulates Ry R receptor-mediated calcium release while promoting mitochondria to produce ATP,and maintains calcium pump and the concentration of intracellular calcium ions thus exerts a biological function.This study aimed to study the therapeutic effect of caffeine on DCP in rats,and to explain its possible mechanism of function,in order to explore a new approach to DCP therapy.Part I Effect of caffeine on the function of diabetic bladder in ratsObjective:To investigate the effect and possible mechanism of caffeine on the function improvement of DCP in rats.Methods:Sprague–Dawley rats(n=64;200–220 g)were purchased from Nanjing Medical University Animal Experiment Center.The 40 rats received intraperitoneal injections of 60 mg/kg streptozotocin(STZ),whereas the other 24 rats received the same volume of citric acid buffer.Fasted blood glucose was analyzed 72 h after STZ injection;a level > 16.7 mmol/L was considered to indicate successful induction of diabetes.And then we chosed 24 diabetic rats as DM group and DM+caffeine group(n=12),24 untreated rats as control group and caffeine group(n=12).The rats of DM+caffeine group and caffeine group were treated with caffeine by oral gavage(10mg/Kg/d)for 16 weeks.Rats in the control group and DM group were orally administered with the same amount of citrate buffer per day.Rat body weight,FBG,urination time,urination interval,maximum bladder urinary pressure and bladder wet weight were measured.Then the rats were sacrificed,and the Dorsal root ganglion(DRG)and bladder tissues were taken.The ELISA technique was used to detect the level of nerve growth factor(NGF),brain-derived neurotrophic factor(BDNF)and calcitonin gene related peptide(CGRP)in the DRG and bladder tissue.The apoptotic cells in DRG were detected by TUNEL method.RT-PCR,Western blot and immunohistochemistry were used to detect the expression of CGRP m RNA and protein in DRG.The apoptotic cells in bladder tissues were detected by TUNEL method.The expressions of Bcl-2,Bax and ?-SM-actin m RNA and protein in bladder tissues were detected by RT-PCR and Western blot.The expression of CGRP receptor(calcitonin gene related peptide receptor,CGRPR)in bladder tissue was detected by immunohistochemistry.Results:1.General condition of the rats and urinary function of the bladder: 40 rats were prepared with STZ in DM model,31 were successful,and the success rate was77.50%.There was no significant difference in body weight between the control group and the caffeine group(P>0.05).Compared with the control group and the caffeine group,the body weight of the DM group and the DM+caffeine group decreased significantly,the decrease was more obvious in DM group,and the difference between the groups was statistically significant(P<0.05).Compared with the control and caffeine group,the difference of FBG was not significan(P>0.05).There was no significant difference in FBG between the DM group and the DM+caffeine group(P>0.05).Compared with the control group and the caffeine group,the FBG of the rats in the DM group AND DM+caffeine group was significantly increased,and the difference was statistically significant(P<0.05).There was no significant difference in the urinary function between the control group and the caffeine group(P>0.05).Compared with the control and caffeine group,the urinary function of the DM group and the DM+caffeine group decreased,but the urinary function of the DM+caffeine group was better than that of the DM group.The difference was statistically significant(P<0.05).2.Apoptosis in rat DRG: TUNEL test showed that there were almost no apoptotic cells in DRG of control group and caffeine group,and more apoptosis was observed in DRG of DM group and DM+ caffeine group.Cells,but the apoptotic cells in the DRG of the DM+caffeine group were less than the DM group,and the difference was statistically significant(P<0.05).3.Levels of NGF and BDNF in rat DRG: ELISA results showed that there was no significant difference in the levels of NGF and BDNF between the control group and the caffeine group(P>0.05).Compared with the control and caffeine group,the levels of NGF and BDNF in DRG of DM group and DM+caffeine group were decreased,but the levels of NGF and BDNF in DRG of DM+caffeine group were higher than those of DM group,and the difference was statistically significant(P<0.05).4.CGRP levels in rat DRG: RT-PCR results showed that CGRP m RNA levels in DRG of DM group and DM+ caffeine group were significantly lower than those of control group and caffeine group,but the level of CGRP m RNA in DRG of DM+caffeine group rats was higher than that of DM group(P<0.05).The results of Western blot showed that the relative expression of CGRP protein in DRG of DM group and DM+caffeine group was significantly lower than that of control group and caffeine group,the relative expression of CGRP protein in DRG of DM+caffeine group was higher than that in DM group,the difference was statistically significant(P<0.05).The results of immunohistochemistry showed that the positive rate of CGRP in DRG of DM group was significantly lower than that of the control group,caffeine group and DM+caffeine group,the difference was statistically significant(P<0.05).The positive rate of CGRP in DRG of DM+caffeine group was lower than that of the control group and caffeine group,but the difference between the groups was not statistically significant(P>0.05).5.NGF,BDNF and CGRP levels in rat bladder tissue: ELISA results showed that there was no significant difference in the levels of NGF,BDNF and CGRP in bladder tissue between control group and caffeine group(P>0.05).Compared with the control group and caffeine group,the levels of NGF,BDNF and CGRP in the bladder tissue of the DM group and the DM+caffeine group were decreased,but the levels of NGF,BDNF and CGRP in the bladder tissue of the DM+caffeine group were higher than those of the DM group,the difference was statistically significant(P<0.05).6.CGRPR levels in rat bladder tissue: Immunohistochemistry results showed that the positive rate of CGRPR in bladder tissue of DM group was significantly lower than that of control group,caffeine group and DM+caffeine group,the difference between the groups was statistically significant(P <0.05).The positive rate of CGRPR in bladder tissue of DM+caffeine group was lower than that of control group and caffeine group,but there was no significant difference between the groups(P>0.05).7.Apoptosis in rat bladder tissue: TUNEL assay showed that there were almost no apoptotic cells in the bladder tissue of the control group and the caffeine group,and more in the bladder tissue of the DM group and the DM+caffeine group.The apoptotic cells in the bladder tissue of the DM+ caffeine group were less than the DM group,and the difference was statistically significant(P<0.05).8.Bcl-2 expression in rat bladder tissue: RT-PCR results showed that there was no significant difference in Bcl-2 m RNA levels between the control group and caffeine group(P>0.05).Compared with the control group and caffeine group,the Bcl-2m RNA level in the bladder tissue of the DM group and the DM+caffeine group was significantly lower,but the Bcl-2 m RNA level in the bladder tissue of the DM+caffeine group was higher than that of the DM group,the difference was statistically significant(P<0.05).Western blot analysis showed that there was no significant difference in the expression of Bcl-2 protein in the bladder tissue between the control group and the caffeine group(P>0.05).Compared with the control group and the caffeine group,the relative expression of Bcl-2 protein in the bladder tissue of DM group and DM+caffeine group was significantly decreased,but the relative expression of Bcl-2 protein in bladder tissue of DM+caffeine group was higher than that in DM group,the difference was statistically significant(P< 0.05).9.The expression of Bax in rat bladder tissue: RT-PCR results showed that the level of Bax m RNA in bladder tissue of DM group was significantly higher than that of control group,caffeine group and DM+caffeine group.(P<0.05).The level of Bax m RNA in bladder tissue of DM+caffeine group was higher than that of control group and caffeine group,but there was no significant difference between the groups(P>0.05).The results of Western blot showed that the level of Bax protein in bladder tissue of DM group was significantly higher than that of control group,caffeine group and DM+caffeine group,the difference was statistically significant(P<0.05).The level of Bax protein in bladder tissue of DM+caffeine group rats was higher than that in the control group and caffeine group,there was no significant difference between the groups(P>0.05).10.The expression level of ?-SM-actin in rat bladder tissue: RT-PCR results showed that there was no significant difference in ?-SM-actin m RNA levels between the control group and caffeine group(P>0.05).Compared with the control group and the caffeine group,the level of ?-SM-actin m RNA in the bladder tissue of the DM group and the DM+caffeine group was significantly decreased,but the ?-SM-actin m RNA level in the bladder tissue of the DM+caffeine group was higher than the DM group,and the difference was statistically significant(P<0.05).Western blot analysis showed that there was no significant difference in the relative expression of?-SM-actin protein between the control group and the caffeine group(P>0.05).Compared with the control group and the caffeine group,the relative expression of?-SM-actin protein in bladder tissue of DM group and DM+caffeine group was significantly decreased,but the relative expression of ?-SM-actin protein in bladder tissue of DM+caffeine group was higher than that in DM group(P <0.05).Conclusion:1.STZ can successfully induce DM model in rats.The urinary function of bladder in DM rats is decreased.Caffeine can improve the function of diabetic bladder in rats.The levels of NGF,BDNF and CGRP in DRG and bladder tissues of DM rats are decreased.Caffeine can promote its recovery to a certain extent.2.Apoptotic cells appeared in DRG of DM rats.Caffeine can protect cells from GRG from apoptosis to a certain extent.The level of apoptosis gene Bax in bladder tissue of DM rats increased,the level of anti-apoptotic gene BCL-2 decreased,and apoptotic cells increased,caffeine can reverse this situation to a certain extent.3.The levels of CGRPR and ?-SM-actin in bladder tissue of DM rats decreased,and caffeine can promote the expression of CGRPR and ?-SM-actin in bladder tissues of DM rats.4.Caffeine may improve the bladder function of DM rats through the protective effects of both nerve and smooth muscle,which may involve at least CGRP/CGRPR-related signaling pathways.Part II The effect of caffeine on rat DRG neurons and the effect of CGRP on bladder smooth muscle cellsObjective:To investigate the effects of caffeine on rat DRG neurons(DRGN)and CGRP on bladder smooth muscle cells(BSMC),and to analyze the possible mechanism of caffeine on the improvement of diabetic bladder function in rats.Methods:12SD rats(200-220 g)were acquired from the Animal Experimental Center of Nanjing Medical University.Post anesthetization of SD rats,DRG and bladder were taken,and primary culture of DRGN,subculture of BSMC and identification were performed.DRGN primary culture was divided into control group,caffeine group,high glucose group,high glucose+caffeine group,control group with 10% FBS DMEM / F12 culture medium,caffeine group with 4 m M caffeine,10% FBS DMEM/ F12 culture medium,high glucose group with 25 m M glucose,10% FBS DMEM /F12 culture medium,high glucose+caffeine with 25 m M glucose,4 m M caffeine and10% FBS DMEM/F12 culture medium.The Fura-2/AM fluorescent probe was used to detect changes in intracellular calcium concentration in DRGN.The concentrations of NGF,BDNF and CRGP in the DRGN culture medium were determined by ELISA.The vitality of DRGN cells was detected by MTT assay.The TUNEL method was used to detect the apoptosis of DRGN.Expression of CGRP m RNA and protein in DRGN was determined by RT-PCR and Western blot.The third generation BSMC were further divided into control group,CGRP group,high glucose group and high glucose+CGRP group,where they were added in a DMEM culture medium containing :10% FBS,4 Mm CGRP and 10% FBS,25 m M glucose and 10% FBS,25 m M glucose,4 m M CGRP and 10% FBS respectively.The activity of DRGN cells was detected by MTT assay.The apoptosis of BSMC was detected by TUNEL method.The proliferation of BSMC was analyzed by Ki67 immunofluorescence and Flow cytometry.RT-PCR,Western blot and immunofluorescence were used to detect the expression of ?-SM-actin m RNA and protein in BSMC cells,as well as expression of ERK and P-3 in bladder tissue.Results:1.Identification of DRGN cells cultured in vitro: Using sensory neuron specific antibody NSE immunofluorescence to detect the cultured cells,more NSE+neurons could be seen under microscope,the neurons body were oval or round,most of them were pseudounipolar neurons,and some of them were bipolar neurons.2.Changes of calcium ion concentration in DRGN cells in vitro: Fura-2/AM fluorescent probe detection showed that the difference in resting calcium ions in DRGN cells of control group,caffeine group,high glucose group,high glucose+caffeine was not statistically significant(P>0.05).Compared with the control group and the caffeine group,the peak calcium ion in the DRGN cells of the high glucose group and the high glucose+caffeine group was significantly reduced(P<0.05),However the peak calcium ion concentration in the DRGN cells of the high glucose+caffeine group was significantly higher than the high glucose group(P<0.05)3.Activity and apoptosis of DRGN cells cultured in vitro: MTT assay showed that the OD values of DRGN between each group was not statistically significant(P>0.05).Moreover TUNEL test results showed that there were no significant differences in apoptotic cells among the groups(P>0.05).4.The levels of NGF and BDNF in DRGN culture medium: No significant differences were seen in the levels of NGF and BDNF between the control group and the caffeine group(P>0.05).Compared with the control group and the caffeine group,the levels of NGF and BDNF in the DRGN medium of the high glucose group and the high glucose+caffeine group were significantly decreased,but the levels of NGF and BDNF in the DRGN medium of the high glucose+caffeine group were higher than that in the high glucose group,the difference was statistically significant(P <0.05).5.The level of CGRP in DRGN cultured in vitro: Compared with control group and caffeine group,the CGRP level in the DRGN medium of high glucose and high glucose+caffeine group was significantly decreased,but the CGRP level in the high glucose+caffeine group was higher than that in the high glucose group(P<0.05).Additionally the levels of CGRP m RNA and protein in high glucose group and high glucose+caffeine group were significantly decreased,as compared with the control and caffeine group.However the level of CGRP m RNA in high glucose+caffeine group was much higher than that in high glucose group(P<0.05).6.Activity and apoptosis of BSMC cultured in vitro: MTT assay showed that there were no significant differences in OD values among all groups(P>0.05).TUNEL results demonstrated that there were almost no apoptotic cells in all groups.7.Proliferation of BSMC cells in vitro: Ki67 immunofluorescence and Flow cytometry analysis established that there was no difference in both number of Ki67+ cell and cell proliferation index between each group(P>0.05)8.Expression of ?-SM-actin in BSMC cultured in vitro: Immunofluorescence results showed that the ?-SM-actin structure in the high glucose group was disordered whereas in the other 3 groups the structure was clear.There was a decreased m RNA and protein levels of ?-SM-actin in the high glucose and the high glucose+CGRP group when compared to the control and CGRP group.On the contrary the m RNA and protein levels of ?-SM-actin in high glucose+CGRP group were significantly higher than that in the high glucose group(P<0.05).9.The levels of p38 and ERK in BSMC cultured in vitro: Levels of p-p38 and p-ERK protein in the high glucose and the high glucose+CGRP group were markedly low in contrast to levels in the control and CGRP group.Nevertheless the levels of p-p38 and p-ERK in the high glucose+CGRP group were higher than that in the high glucose group(P<0.05).Conclusion:1.In vitro high glucose environment do not induce apoptosis of rat DRGN,but inhibited the expression of NGF,BDNF and CGRP in DRGN.Caffeine can promote its recovery to a certain extent,which may be related to the regulation of intracellular calcium ion concentration by caffeine.2.In vitro high glucose environment do not induce apoptosis of rat BSMC,do not affect the proliferation of BSMC,but inhibit the expression of ?-SM-actin,CGRP can promote its recovery to a certain extent.3.P38 and ERK signaling pathways in rat BSMC are inhibited in high glucose environment in vitro,and CGRP can promote its recovery to a certain extent.4.Caffeine has a protective effect on DRGN in high glucose environment,and promotes the expression of CGRP.The latter can regulate the intracellular P38 and ERK signaling pathways of BSMC and promote the expression of ?-SM-actin in high glucose environment in vitro.