Based On "Method Of Relieving Lung For Dieresis" To Investigate How Yuebi Decoction Regulate The Unstable Bladder Model Of Enuresis

ObjectiveIn this study,we aim to take the Interstitial cells of Cajal(ICC),and takes endoplasmic reticulum stress(ERS)-unfolded protein response as the breakthrough point,uncover the nature and the related mechanism about that Yuebi decoction can down-regulate number of bladder ICC to improve bladder unstable-state resulted by nocturia.Meanwhile it will make a scrupulous demonstration for “method of Relieving lung for dieresis”from modern experimental science.In this study,we aim to figure out the molecular mechanism of the therapeutic effects of Yue-Bi-Tang(YBT)on improving bladder unstable-state resulted by nocturia,via regulating proliferation and apoptosis of Interstitial cells of Cajal(ICC)and establish the basic theory of “Ti-Hu-Jie-Gai” in modern scientific way.MethodExperiment one: Firstly,theproximal urethra ligation method was used to establish the bladder outlet obstruction(BOO)model.and then ICCs were isolated from bladder of model rats.Isolated ICCs were maintained in appropriate medium for several weeks and stored in liquid nitrogen for long term keep.60 BOO model wister rats were randomly divided into 6 groups named as follows:(1)YBTGF group;(2)YBT high-dose group;(3)YBT middle-dose group;(4)YBT low-dose group;(5)Suo-Quan-Wan group and(6)distilled water group(CTL).All rats were orally administered with described medicine,respectively.The dosage of the medicated decoctions was 20 ml/kg body weight,wich was equal to 5.0 grams of formula per kg body weight for 14 days.All rats were administrated and blood from the rats was collected and centrifuged(3000g for 10min)to obtain the serums.Experiment two:After getting medicated serum and control serum,isolated ICCs described above were co-incubated with different medicated serum,respectively.After certain time,ICCs cells were performed for proliferating,apoptotic or cellular death assays.Ed U staining,TUNEL and CFSE/PI were employed separately for the detections.Experiment three: For determing the effects of different medicated-serum on endoplasmic reticulum stress(ERS),the pro-apoptotic and pro-survival branches of signal pathways were detected,including ATF6,GRP78,CHOP,caspase-12 and JNK.Experiment four:Then Fluo 3-AM probe was used for detecting calcium ion in ICCs after medicated-serum treatment and imaged using fluorescence microscope for measuring the concentration of intracellular calcium ion.Result1.Isolation,incubation and characterization of ICCs from OOD model rats.24 hours after isolation of ICCs from bladder of modeled rats,adherent cells were observed.It is observed that the cell morphology was consistent with standard image,For further confirming the characterization of ICCs,fluorescent immunoblotting was performed to detect the marker protein,c-Kit.As expected,most of cells isolated exhibited c-kit-positive signal.2.The effects of medicated-serum of cell proliferation:After Ed U staining,it is observed that,with the growing dose of YBT-medicated serum,the proliferating capacity of ICCs decreased rapidly.GF-medicated serum presentd a similar effect on proliferating capacity on ICCs with low-dose YBT-medicated serum;while SQW-medicated serum exhibited a similar effect on proliferation with high-dose YBT-medicated serum.3.The effect of medicated-serum on apoptosis in ICCs With the grow dose of YBT-medicated serum,the apoptotic rate of ICCs grow simultaneously,while control serum showed no effect on apoptosis in ICCs.SQW and GF-medicated serum showed slight apoptotic-promoting ability.4.Pro-survival branch signal pathway Being detected by m RNA and protein levels,the hallmarks of pro-survival signal pathway,ATF6 and GRP78 was upregulated by YBT-medicated serum(P<0.05).High-dose of YBT-medicated serum presented more obvious upregulation on these genes.By compared with CTL group,SQW and GF-medicated serum also showed promoting effects on these two genes.(P<0.05)5.the effects of medicated serum on pro-apoptotic branch of signal pathway By detecting the hallmarks of pro-apoptotic signal pathway,the m RNA and protein levels of JNK,CHOP,caspase were detected separately and the results showed that,by compared with CTL group,YBT failed to modulate the expressing levels of these genes on both m RNA and protein levels(P>0.05).6.The effect of medicated serum on calcium ion in ICCs All medicated serum failed to induced the concentration of calcium ion in ICCs.Conclusions1.Although Yuebi decoction can significantly inhibiting the proliferation of ICCs.cajal cells proliferation,while failed to promoting apoptosis.2.Yuebi decoction can inhibit the proliferation of ICCs.regulate bladder unstable-state and result enuresis.The potential mechanism is described as follows:YBT medicated serum promoting ERS by inducing the expressing levels of ATF6,GRP78,but not pro-apoptotic signal pathway.The induction of ERS hallmarks contributes to the survival of ICCs.3.It is approved that “method of Relieving lung for dieresis”is functional to c ure enuresis.