Long-pulse gastric electrical stimulation protects interstitial cells of Cajal in diabetic rats via IGF-1 signaling pathway

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1 Submit Mnusript: Help Desk: DOI: /wjg.v22.i World J Gstroenterol 2016 June 21; 22(23): ISSN (print) ISSN (online) 2016 Bishideng Publishing Group In. All rights reserved. ORIGINAL ARTICLE Bsi Study Long-pulse gstri eletril stimultion protets interstitil ells of Cjl in dibeti rts vi IGF-1 signling pthwy Hi Li, Yn Chen, Shi Liu, Xio-Hu Hou Hi Li, Yn Chen, Shi Liu, Xio-Hu Hou, Division of Gstroenterology, Union Hospitl, Tongji Medil College, Huzhong University of Siene nd Tehnology, Wuhn , Hubei Provine, Chin Author ontributions: Liu S oneived nd designed the experiments; Li H nd Chen Y performed the experiments; Li H nlyzed the dt; Liu S nd Hou XH ontributed regents/ mterils/nlytil tools; Li H wrote the pper; nd Hou XH is the min ontroller of the lbortory. Supported by Ntionl Nturl Siene Foundtion of Chin, No nd No Institutionl review bord sttement: This study ws reviewed nd pproved by the Institutionl Review Bord of Union Hospitl, Tongji Medil College, Huzhong University of Siene nd Tehnology, Chin. Institutionl niml re nd use ommittee sttement: All experiments involving nimls were reviewed nd pproved by the Animl Cre nd Use Committee of Tongji Medil College, Huzhong University of Siene nd Tehnology (IACUC No. 488). Conflit-of-interest sttement: We delre tht the uthors of this rtile do not hve ny onflit of interest. Dt shring sttement: No dditionl dt re vilble. Open-Aess: This rtile is n open-ess rtile whih ws seleted by n in-house editor nd fully peer-reviewed by externl reviewers. It is distributed in ordne with the Cretive Commons Attribution Non Commeril (CC BY-NC 4.0) liense, whih permits others to distribute, remix, dpt, build upon this work non-ommerilly, nd liense their derivtive works on different terms, provided the originl work is properly ited nd the use is non-ommeril. See: lienses/by-n/4.0/ Correspondene to: Shi Liu, MD, Professor, Division of Gstroenterology, Union Hospitl, Tongji Medil College, Huzhong University of Siene nd Tehnology, 1277 Jiefng Avenue, Wuhn , Hubei Provine, Chin. shiliugo@yhoo.om Telephone: Fx: Reeived: Mrh 1, 2016 Peer-review strted: Mrh 3, 2016 First deision: April 1, 2016 Revised: April 14, 2016 Aepted: My 4, 2016 Artile in press: My 4, 2016 Published online: June 21, 2016 Abstrt AIM: To investigte the effets of different prmeters of gstri eletril stimultion (GES) on interstitil ells of Cjl (ICCs) nd hnges in the insulin-like growth ftor 1 (IGF-1) signl pthwy in streptozotoinindued dibeti rts. METHODS: Mle rts were rndomized into ontrol, dibeti (), dibeti with shm GES ( + SGES), dibeti with GES1 (5.5 pm, 100 ms, 4 ma) ( + GES1), dibeti with GES2 (5.5 pm, 300 ms, 4 ma) ( + GES2) nd dibeti with GES3 (5.5 pm, 550 ms, 2 ma) ( + GES3) groups. The expression levels of -kit, M-SCF nd IGF-1 reeptors were evluted in the gstri ntrum using Western blot nlysis. The distribution of ICCs ws observed using immunolbeling for -kit, while smooth musle ells nd IGF-1 reeptors were identified using α-sma nd IGF-1R ntibodies. Serum level of IGF-1 ws tested using enzyme-linked immunosorbent ssy. RESULTS: Gstri emptying ws delyed in the group but improved in ll GES groups, espeilly in the GES2 group. The expression levels of -kit, M-SCF nd IGF-1R were deresed in the group but inresed in ll GES groups. More ICCs (-kit + ) nd smooth musle ells (α-sma + /IGF-1R + ) were observed in ll GES groups thn in the group. The verge level WJG June 21, 2016 Volume 22 Issue 23

2 Li H et l. Mehnisms by whih long-pulse GES ffets interstitil ells of Cjl of IGF-1 in the group ws mrkedly deresed, but it ws up-regulted in ll GES groups, espeilly in the GES2 group. CONCLUSION: The results suggest tht long-pulse GES promotes the regenertion of ICCs. The IGF-1 signling pthwy might be involved in the mehnism underlying this proess, whih results in improved gstri emptying. Key words: Long-pulse gstri eletril stimultion; Interstitil ells of Cjl; Gstri emptying; IGF-1 pthwy; Dibetes mellitus; Gstrointestinl motility disorders The Author(s) Published by Bishideng Publishing Group In. All rights reserved. Core tip: Gstri eletril stimultion (GES) regultes gstri motility nd protets the interstitil ells of Cjl (ICCs). However, the mehnisms underlying these proesses hve not been determined. In this study, we report tht long-pulse GES with three different prmeters proteted ICCs nd tht the insulin-like growth ftor 1 signling pthwy is probbly involved in this proess. One GES prmeter (5.5 pm, 300 ms, 4 ma) showed immense potentil s linil pplition for pplying long-pulse GES s the optiml prmeter. Li H, Chen Y, Liu S, Hou XH. Long-pulse gstri eletril stimultion protets interstitil ells of Cjl in dibeti rts vi IGF-1 signling pthwy. World J Gstroenterol 2016; 22(23): Avilble from: URL: om/ /full/v22/i23/5353.htm DOI: org/ /wjg.v22.i INTRODUCTION Gstrointestinl motility disorders (suh s erly stiety, bloting nd vomiting) often plgue ptients with longstnding dibetes. However, the pthogenesis of these disorders remins unler, nd effetive phrmologil mngement is limited for these symptoms. Fortuntely, few reent non-phrmologil tretments, inluding gstri eletril stimultion (GES), hve renewed the hope for hieving new mngement strtegies, but the mehnisms underlying their effiy remin unler. Reently, number of studies hve indited tht interstitil ells of Cjl (ICCs) my ply n importnt physiologil role in oordinting gstri ontrtile tivity nd gstri motility [1,2]. C-kit protein is known to lolize on the surfe of ICCs nd to be tivted by intertions between kit reeptors nd stem ell ftor (SCF). SCF/-kit signling is importnt for mintining the ICC phenotype nd ICC prolifertion, differentition nd survivl [3,4]. Furthermore, some studies hve suggested tht dmge to or the loss of ICCs leds to gstrointestinl motility disorders in dibeti models [5-7] nd tht dereses in the levels of SCF nd insulin-like growth ftor 1 (IGF-1) re involved in ICC defiieny [8]. Aording to the results of reent studies, SCF nd IGF-1, whih re neessry for the differentition nd mintenne of the ICC phenotype, re derived from smooth musle ells (SMCs), nd IGF-1 inreses the expression of SCF [9]. Moreover, the IGF-1 reeptor (IGF- 1R) ppers to be lking in mture ICCs, nd IGF-1 is likely to diretly intert with reeptors on SMCs to enhne SCF synthesis, resulting in protetive effet in ICCs [8]. Bsed on reent study, the levels of serum IGF-1 nd its binding protein 3 re ltered in type 1 dibetes nd tht irulting IGF-1 nd its binding protein 3 ontrol oloni stem ell funtion nd gstrointestinl omplitions of dibetes [10]. Generlly, GES is lssified into three tegories ording to the length of the pulse nd the energy tht is pplied: the short-pulse GES, the long-pulse GES nd the dul-pulse GES. The effets of GES vried with the prmeters. In refrtory gstropresis ptients who nnot undergo drug therpy due to serious side effets or who re unble to undergo surgil tretment, GES is n lterntive option. Our previous study indited tht GES might inrese the expression of membrne stem ell ftor (M-SCF) in streptozotoin (STZ)-indued dibeti rts [11]. However, the mehnism by whih GES ffets gstri motility nd the involvement of IGF-1 signling remin unknown. The im of this study ws to: (1) systemtilly ssess the effets of pplying GES with different prmeters on gstri emptying; (2) sertin the effets of GES on ICCs in dibeti rts; nd (3) determine whether the IGF-1 pthwy is involved in the regenertion of ICCs. MATERIALS AND METHODS Animl subjets Adult mle Sprgue-Dwley rts ( g) obtined from the Experiment Animl Center of Tongji Medil College were used in this study. All pproprite mesures were dopted to minimize disomfort or pin to the nimls. The rts were housed in stndrdized lbortory onditions ( 12/12 h light/drk yle t 22 ) nd llowed free ess to stndrd solid food nd sterile wter. The rts were formlly enrolled fter they were dpted to the lbortory onditions for one week. All experiments were reviewed nd pproved by the Institutionl Animl Cre nd Use Committee of Tongji Medil College. Experimentl protools The nimls were rndomized into one of six equl groups (6 rts/group): ontrol, dibeti (), dibeti nd shm GES ( + SGES), dibeti nd GES prmeter 1 (5.5 pm, 100 ms, 4 ma) ( + GES1), dibeti nd GES prmeter 2 (5.5 pm, 300 ms, 4 ma) ( + GES2), nd dibeti nd GES prmeter 3 (5.5 pm, 550 ms, 2 ma) ( + GES3). Dibetes ws WJG June 21, 2016 Volume 22 Issue 23

3 Li H et l. Mehnisms by whih long-pulse GES ffets interstitil ells of Cjl Surgery STZ Model Srified Gstri eletril stimultion -3 wk -1 wk 0 wk 2 wk 4 wk 6 wk Weight nd gluose (1) (2) (3) (4) (5) Figure 1 Shemti representtion of the gstri eletril stimultion study protool. After surgery of eletrode implnttion, the rts hd 2 wk to reover from the surgery. After streptozotoin injetion, the dibeti rts were rndomized into four lrge groups depending on the prmeter of gstri eletril stimultion (GES): (dibeti group) + SGES (dibeti with shm GES), + GES1 (GES prmeter 1), + GES2 nd + GES3 groups for 6 wk. GES ws dopted for 30 min/d, 7 d/wk during the whole proess of the experiment. The body weights nd blood gluose levels were mesured before the injetion of STZ nd during weeks 0, 2, 4, nd 6 fter the indution of dibetes. indued using STZ (60 mg/kg, ip, Alexis Biohemil, United Sttes). Rts in the ontrol group were injeted with equl volume of diluent. Blood gluose onentrtions were mesured one week fter the injetion. The rts were onsidered dibeti if their blood gluose level inresed nd ws mintined t equl to or more thn 16.7 mmol/l. Body weight nd blood gluose levels were lso tested before the rts were injeted nd during weeks 0, 2, 4, nd 6 fter the indution of dibetes. After sequentil GES intervention ws performed for 6 wk, gstri emptying ws mesured in ll groups. Serum smples were olleted for enzyme-linked immunosorbent ssy (ELISA), nd the rts were srified nd the speimens of the ntrum were quired. Eh ntrum speimen ws seprted into two piees. One of these ws stored t -80 for Western blot nlysis, nd reltively lrge piee ws fixed in Zmboni s fixtive [1.5% sturted piri id solution nd 2% prformldehyde in 0.1 M phosphte buffer solution (PBS, ph = 7.4)] for the immunofluoresene study. Gstri eletril stimultion Rts in the GES nd shm GES groups were nesthetized using pentobrbitl sodium (30 mg/kg, ip, Sigm, United Sttes). A midline lprotomy ws performed, nd stimulting eletrodes were piered the subseros of the stomh nd pled on the serosl surfe long the greter urvture. The distl pir of eletrodes ws bout 0.5 m wy from the pylorus nd the proximl pir ws pproximtely 1.5 m wy from the distl pir. The eletrodes of the proximl pir were pled 0.3 m prt. The wires were tunneled through the nterior bdominl wll nd led out of the skin so they ould be onneted to the stimultor (G6805-2A; Shnghi Huyi Medil Instrument Ftory, Chin). The bdominl wll ws losed using simple interrupted suture. After the rts reovered from the surgery ompletely (usully 2 wk), they ontinuously reeived GES intervention for 30 min/dy for 6 wk (Figure 1). The following respetive long-pulse GES frequenies, pulse widths nd mplitude prmeters were used: GES1, 5.5 yles/min (pm), 100 ms nd 4 ma; GES2, 5.5 pm, 300 ms nd 4 ma; nd GES3, 5.5 pm, 550 ms nd 2 ma. These prmeters were shown to be effetive nd representtive in previous experiments [11-13]. Rts in the + SGES group were onneted only to the stimultor nd were not stimulted with n eletri urrent. Mesurement of gstri emptying The test mels, inluding rboxymethylellulose (15 mg/ml) nd phenol red (0.5 mg/ml), were ontinully stirred nd mintined t 37. After the rts were deprived of food overnight, the nimls were fed 2 ml of the test mel using stright gvge needle. After 30 min, the rts were quikly srified using ervil dislotion. The stomh ws quired fter it ws ligted t the pylorus nd rdi, nd then it ws opened. The gstri ontents were pled into test tube nd then wshed using distilled wter. A NOH solution (20 ml, 1 mol/l) ws pled in eh test tube. Absorbne t 560 nm ws red using spetrophotometer (U-2900, Hithi, Jpn) to determine the quntity of remined phenol red. Gstri emptying ws lulted using the following formul: gstri emptying (%) = 100 (1-X/Y), where X is the bsorbne of phenol red mesured t 30 min fter the test mel, nd Y is the bsorbne of phenol red in the ontrol rts tht were srified immeditely fter they were fed the test mel [14]. WJG June 21, 2016 Volume 22 Issue 23

4 Li H et l. Mehnisms by whih long-pulse GES ffets interstitil ells of Cjl Immunofluoresene studies The gstri ntrum tissues were rpidly fixed in Zmboni s fixtive for 24 h t room temperture. They were then dehydrted, embedded in prffin nd ut t thikness of 5-7 μm. The prffin setions were deprffinized nd hydrted before ntigen retrievl ws performed. Endogenous peroxidse ws ontrolled using 3% hydrogen peroxide (H2O2) for 30 min. The setions were then mirowved (750 W) for 5 min, nd nonspeifi retions were inhibited using norml got serum for 20 min. The primry ntibodies, inluding IGF-1R (1:200; Snt Cruz Biotehnology, In), -kit (1:150; Snt Cruz Biotehnology, In) nd α-sma (1:500; Snt Cruz Biotehnology, In), were dded dropwise to the prffin setions whih were pled t 4 overnight. The setions were then rinsed three times using PBS (PH 7.4) nd inubted in seondry ntibodies for 60 min t 37, followed by inubtion with DAPI (Sigm, United Sttes) for 30 min. The setions were then ounterstined nd dehydrted. The speimens were observed using lser snning onfol mirosope (Olympus, Tokyo, Jpn). Western blot nlysis The ntrum speimens were homogenized in RIPA buffer (Upstte, United Sttes) ontining protese inhibitor (Beyotime, Chin) nd inubted for 30 min on ie. The liquid ws entrifuged t g t 4 for 8 min, nd the superntnt ontining the totl extrted proteins ws then olleted. BCA regent (Piere, Rokford, IL, United Sttes) ws used to nlyze the protein onentrtion of eh smple. Totl proteins were seprted using 10% sodium dodeyl sulfte-polyrylmide gel eletrophoresis nd then trnsferred to PVDF membrnes (Millipore, United Sttes). The membrnes were immersed in 5% skim milk solution for 1 h nd then inubted with primry ntibodies ginst -kit (1:200, Sigm-Aldrih, United Sttes), SCF (1:200, Abm, United Kingdom), IGF-1R (1:200, Boster, Chin) or β-tin (1:1000, Beyotime, Chin) t 37 with gentle shking for 1 h, followed by mintenne t 4 overnight. After inubtion with seondry ntibodies for 1 h, the bnds on the PVDF membrnes were visulized using enhned hemiluminesene (Amershm Phrmi, United Sttes). A densitometry nlysis ws onduted using AlphView softwre. ELISA For this experiment, pproximtely 2 ml of blood ws obtined from eh rt to study differenes in serum IGF-1 levels. The onentrtion of IGF-1 ws quntified using rt IGF-1 ELISA kits (RyBioteh, United Sttes). A stndrd urve ws estblished for IGF-1 by testing the stndrd with spetrophotometer t 450 nm, nd the onentrtion of IGF-1 in the serum ws then determined by ompring the optil densities of the study smples to those of the stndrd smples. Eletron mirosopy The gstri ntrum ws immersed in fixtive solution (2.5% glutrldehyde) for 2 h t 4. Tissue smples (pproximtely 1 mm 5 mm) were seprted from the gstri ntrum nd soked in 1% OsO4 for min. They were then rinsed with 0.1 mol/l phosphte buffer nd dehydrted in ethnol. The tissue smples were immersed in propylene oxide followed by mixtures of Epon Resin nd propylene oxide for 2 h, nd then embedded in Epon. An ultrmirotome ws used to identify the regions of interest in the study nd to setion them into ultrthin setions (70 nm). The setions were visulized using trnsmission eletron mirosope (Teni G212, FEI, The Netherlnds). Sttistil nlysis The dt re presented s the men ± SEM. One-wy nlysis of vrine ws used to evlute differenes mong groups. The lest signifint differene post ho test ws pplied to ompre differenes between groups. A P vlue < 0.05 ws onsidered sttistilly signifint. Sttistil nlyses were performed using SPSS 17.0 softwre. RESULTS Weight nd blood gluose level Bseline weight did not mrkedly differ between the groups (Figure 2A). The weights of the rts in the group were mrkedly lower t the end of weeks 2, 4 nd 6 (P = 0.013, P = nd P < , respetively) thn the weights of the ontrols. The weights of the + GES1, + GES2 nd + GES3 groups were signifintly higher t the 6th week thn the weights in the group (P = 0.035, nd 0.031, respetively). As shown in Figure 2B, bseline blood gluose levels were not signifintly different between the groups. After the indution of dibetes, the blood gluose levels were higher in the group thn in the ontrol group for the reminder of the experiment (P < ). After long-pulse GES intervention ws pplied, blood gluose levels were not mrkedly different between the + GES1, + GES2 or + GES3 group nd the group (P = 0.332, nd 0.416, respetively). Eletron mirosopy In the ontrol group, ICCs showed higher eletrondense ytoplsm thn SMCs nd were bundnt in the endoplsmi retiulum, mitohondri nd bsl lmin (Figure 3A). In ddition, they showed lose onnetion with SMCs nd enteri nerves. However, in the group, ICCs were seriously impired (Figure 3B): swollen endoplsmi retiulum nd mitohondri, extensive vuoliztion, ytoplsmi depletion nd lmellr bodies were frequently observed in their ell bodies. The interellulr spes were dilted, WJG June 21, 2016 Volume 22 Issue 23

5 Li H et l. Mehnisms by whih long-pulse GES ffets interstitil ells of Cjl A Body weight (g) b b b b b b b b b -1 W 0 W 2 W 4 W 6 W SGES + GES1 + GES2 + GES3 B Blood gluose (mmol/l) W 0 W 2 W 4 W 6 W 0 + SGES + GES1 + GES2 + GES3 Figure 2 Body weights nd blood gluose levels t different time points in different groups. A: The body weights of the dibeti rts were obviously deresed ompred with the ontrols t 6 wk (P < 0.05). The weights in ll GES groups were inresed ompred with the (dibeti group) (P < 0.05). There ws no signifint differene between the nd + SGES groups (P > 0.05); B: Compred with the ontrol group, the blood gluose level of the group ws signifintly inresed t weeks 0, 2, 4 nd 6 (P < 0.05 for ll). P < 0.05 vs ontrol; b P < 0.05 vs group. gp juntions between ICCs nd enteri nerves were redued nd exudtion of fibrin ws osionlly observed. ICCs in the + SGES group displyed ultrstruturl hnges tht were similr to those in the ICCs in the group (Figure 3C). However, most of the ICCs in the + GES1 (Figure 3D), + GES2 (Figure 3E) nd + GES3 (Figure 3F) groups were repired, nd minor dmge (the struture of the ytomembrne ws reltively omplete, orgnelles were bundnt, there were slight ytoplsmi depletion, slightly swollen endoplsmi retiulum nd mitohondri nd limited vuoliztion, there were few denuded ribosomes nd smll number of lysosomes, nd there ws little heterohromtin.) ws osionlly observed. Gstri emptying Figure 4 shows the effet of GES on gstri emptying. Gstri emptying in the group ws slower thn tht in the ontrol group (P < ). SGES did not mrkedly ffet gstri emptying ompred to the group (P = 0.573), but GES1, GES2 nd GES3 signifintly improved the delyed gstri emptying from 35.89% ± 4.43% to 48.27% ± 2.20%, 61.84% ± 4.87 nd ± 2.26%, respetively (P = 0.03, nd , respetively). These improvements were signifintly different between the + GES1 group nd the + GES2 group (P = 0.029), inditing tht the effet of GES2 on gstri emptying ws stronger thn tht of GES1. Western blot nlysis of -kit, SCF nd IGF-1R As shown in Figure 5A nd B, the expression of -kit ws evluted in the ntrum. The level of -kit ws mrkedly lower in the group thn in the ontrol group (P < ). Conversely, the expression of -kit ws drmtilly higher in the + GES1, + GES2 nd + GES3 groups thn in the group (P = , P < nd P = , respetively). The expression of M-SCF (Figure 5C nd D) ws lower in the group thn in the ontrol group (P < ). However, GES1, GES2 nd GES3 resulted in higher WJG June 21, 2016 Volume 22 Issue 23

6 Li H et l. Mehnisms by whih long-pulse GES ffets interstitil ells of Cjl Tble 1 Serum levels of IGF-1 in eh group IGF-1 (ng/ml) + SGES ± ± ± GES ± GES2,e ± GES ± 9.37 Dt re given s men ± SEM. P < 0.05, vs group; P < 0.05 vs group; ep < 0.05, + GES2 vs + GES1 group. + SGES SMC ICC ICC SMC SMC ICC 2 mm 2 mm + GES1 2 mm + GES2 + GES3 ICC ICC SMC SMC ICC SMC 2 mm 2 mm 2 mm Figure 3 Eletron mirosopy of interstitil ells of Cjl in eh group. In the (dibeti) nd SGES (dibeti with shm GES) groups, interstitil ells of Cjl (ICCs) were mrkedly ffeted ompred with the ontrol group, while they ppered to be lmost norml in struture or hd minor dmge in the (dibeti group) + GES1 (GES prmeter 1), + GES2 nd + GES3 groups. Sle brs = 2 μm. level of expression of M-SCF thn ws observed in the group (P = 0.001, P < nd P < , respetively). The effet of GES2 ws stronger thn tht of GES1 (P = 0.028). Compred to the ontrol, the expression of IGF1R (Figure 5E nd F) ws mrkedly lower in the group (P < ), wheres the level of IGF-1R in the + GES1, + GES2 nd + GES3 groups ws signifintly higher thn the level in the group (P = 0.004, P < nd P = 0.001, respetively). This effet ws stronger in the GES2 group thn in the GES1 group (P = 0.039). groups were ± 9.75, ± 5.61, ± 5.02, ± 6.74, ± 9.97 nd ± 9.37 ng/ml, respetively. The verge IGF-1 level in the group ws lower thn tht in the ontrol group (P < ). However, the IGF-1 level ws higher in the + GES1, + GES2 nd + GES3 groups thn in the group (P < for ll). The level of IGF-1 ws not mrkedly different between the + SGES nd the groups (P = 0.794). Immunofluoresene studies of -kit nd IGF-1R As shown in Figure 6, -kit immunoretivity reveled the distribution of ICCs in the gstri ntrum of eh + group. There were number of -kit ells in the ontrol group. However, few ICCs were observed in the group. Similr results were found in the + SGES group, nd long-pulse GES interventions Serum IGF-1 levels Tble 1 shows the levels of IGF-1 in the serum in eh group. The IGF-1 levels in the,, + SGES, + GES1, + GES2 nd + GES3 WJG June 21, 2016 Volume 22 Issue 23

7 Li H et l. Mehnisms by whih long-pulse GES ffets interstitil ells of Cjl Gstri emptying (%) mrkedly inresed the expression of -kit in the + GES1, + GES2 nd + GES3 groups. In Figure 7, α-sma nd IGF-1R immunoretivity reflets the distribution of smooth musle ells nd IGF-1 reeptors, respetively, in the gstri ntrum of eh group. In the ontrol group, lrge number of IGF-1R + ells ws observed in the intrmusulr lyer, wheres few IGF-1R + ells were observed in the group. Anlogously, few IGF-1R + ells were observed in the + SGES group. However, IGF-1R expression ws mrkedly inresed in the + GES1, + GES2 nd + GES3 groups fter tretment with long-pulse GES. DISCUSSION + SGES Figure 4 Effets of gstri eletril stimultion on gstri emptying. Compred with the ontrol group, gstri emptying in the group ws signifintly delyed (P < 0.01). However, gstri eletril stimultion (GES) signifintly improved the delyed gstri emptying (P < 0.01 for ll). Menwhile, signifine ws found between the (dibeti group) + GES1 (GES prmeter 1) group nd + GES2 group (P = ). P < 0.05 vs ontrol; P < 0.05 vs group; e P < 0.05, + GES1 vs + GES2 group. In the urrent study, pplying GES using vrious settings inresed the expression of M-SCF nd IGF-1/IGF-1R nd improved the regenertion of ICCs in dibeti rts, whih meliorted delyed gstri emptying in these rts. In reent yers, GES hs been proposed s therpeuti option for ptients suffering from refrtory gstropresis. Some studies hve demonstrted tht the effets of GES depend on the GES prmeters. Short-pulse GES improved dyspepti symptoms, while long-pulse GES normlized gstri dysrhythmi nd regulted gstri slow wves, nd dul-pulse GES redued dyspepti symptoms nd gstri dysrhythmi [15-20]. Beuse in some instnes, longpulse GES re-estblishes norml slow wve tivity nd improves gstri emptying, it is usully pplied to tret gstropresis. Bsed on the results of previous studies, frequeny of 5.5 pm, pulse width rnging + GES1 e + GES2 + GES3 from 100 ms to 300 ms nd n mplitude of 4 ma re frequently used nd hve been shown to be effetive in regulting gstri dysrhythmi nd gstri slow wves [21,22]. In this study, we seleted representtive widths of 100 ms (GES1: 5.5 pm, 100 ms, nd 4 ma) nd 300 ms (GES2: 5.5 pm, 300 ms, nd 4 ma) for further study. Additionlly, GES3 (5.5 pm, 550 ms, 2 ma) ws lso demonstrted to be effetive in our previous study [11], nd tht setting ws onsequently lso used in this study. Bellhsène et l [23] demonstrted tht long-pulse GES (7 pm, 300 ms, 4 ma) elerted gstri emptying in nine model of gstropresis. Moreover, MCllum et l [24] disovered tht long-pulse GES (t frequeny tht ws 10% higher thn the intrinsi slow-wve frequeny, 300 ms, 4 ma) elerted gstri emptying in gstropresis ptients. However, Xing et l [20] found tht long-pulse GES (6 pm, 375 ms, 4 ma) did not signifintly influene gstri emptying in nine model. In the present study, delyed gstri emptying ws elerted fter GES intervention, espeilly in the GES2 group (5.5 pm, 300 ms, 4 ma), whih supports the findings of most studies in this field. Our results showed tht long-pulse GES improved delyed gstri emptying in dibeti rts, nd the effet of GES2 (5.5 pm, 300 ms, 4 ma) ws more pronouned thn those of GES1 nd GES3. Thus, the GES2 protool should potentilly be pplied in the lini, but further studies re needed to explore this issue. The mehnisms by whih long-pulse GES ffets gstri emptying remin unler. ICCs hve been shown to ply n importnt role in the regultion of gstri peristlsis, whih signifintly ffets gstri emptying [1,25]. However, few studies hve exmined the involvement of ICCs in the effet of long-pulse GES on gstri emptying. We onduted relted experiments in whih we exmined the effet of GES on ICCs. The results reveled tht long-pulse GES (5.5 pm, 550 ms, 2 ma) resulted in ICC remodeling in dibeti rts. Furthermore, our previous study showed tht both low- nd high-frequeny eletroupunture t ST-36 inresed the number of ICCs [14]. In the present study, GES intervention lso mrkedly reovered the ultrstruture of ICCs to minor injury or norml stte [26] nd inresed the number of ICCs in the ntrum in dibeti rts, inditing tht long-pulse GES might indue ICC remodeling nd further ontribute to improved gstri emptying. The -kit/scf pthwy is one of the most importnt regultors of ICCs. As the lignd of -kit, SCF exists in both soluble form (S-SCF) nd trnsmembrne form (M-SCF). M-SCF my ply more importnt role thn S-SCF during the differentition, survivl nd mintenne of the ICC phenotype [27]. Thus, we foused on M-SCF in our evlution of -kit/scf signling nd our gol of identifying the mehnisms by whih GES results in ICC remodeling. Horváth et l [8] linked redued SCF to n ICC defiieny in dibeti gstropresis nd found tht the level of M-SCF ws WJG June 21, 2016 Volume 22 Issue 23

8 Li H et l. Mehnisms by whih long-pulse GES ffets interstitil ells of Cjl A B kit (145 kd) β-tin (43 kd) +SGES +GES1 +GES2 +GES3 -kit/b-tin SGES + GES1 + GES2 + GES3 C M-SCF (27 kd) +SGES +GES1 +GES2 +GES3 D M-SCF/b-tin e β-tin (43 kd) SGES + GES1 + GES2 + GES3 E IGF-1R (95 kd) β-tin (43 kd) +SGES +GES1 +GES2 +GES3 F IGF-IR/b-tin e SGES + GES1 + GES2 + GES3 Figure 5 Expression levels of relted proteins quntified by Western blot. A, B: The expression of -kit in different groups; C, D: The expression of M-SCF in different groups; E, F: The expression of IGF-1R in different groups. P < 0.05 vs ontrol; P < 0.05 vs (dibeti group) group; e P < 0.05, + GES1 (GES prmeter 1) vs + GES2 group. GES: Gstri eletril stimultion. deresed in the gstri ntrum in dibeti mie. Our results lso indite tht the expression of M-SCF is mrkedly deresed in dibeti rts. However, the effets of GES on the expression of M-SCF hve rrely been investigted. In previous study, we showed tht both low- nd high-frequeny eletroupunture t ST-36 inresed the expression of M-SCF in dibeti rts [14]. In the present study, we found tht GES tretment lso inresed the expression of M-SCF, nd this inrese ws stronger when GES2 (5.5 pm, 300 ms, 4 ma) ws pplied. Therefore, M-SCF is likely to be involved in ICC remodeling s result of long-pulse GES. IGF-1 signling is one of the min ftors tht regulte the expression of M-SCF. IGF-1R, whih is neessry for the differentition nd mintenne of ICC phenotype, is loted on smooth musle ells but not on ICCs [8,28]. Horváth et l [29] showed the first in vitro evidene tht IGF-1 signling ws responsible for mintining the ICC network. In their subsequent study, they suggested tht endogenous IGF-1 expression ws deresed in the dibeti group nd tht dding IGF-1 to the medium improved the expression of M-SCF [9]. The results of the present study show tht the expression of IGF-1R in gstri ntrl SMCs ws mrkedly deresed in the group, WJG June 21, 2016 Volume 22 Issue 23

9 Li H et l. Mehnisms by whih long-pulse GES ffets interstitil ells of Cjl -kit DAPI Merge -kit A B C D E F DAPI Merge Figure 6 Confol mirogrphs of interstitil ells of Cjl. Interstitil ells of Cjl (ICCs) were lbelled with -kit (green) nd DAPI (blue). ICCs were bundnt in the ontrol group (A). Few ICCs were observed in the group (B). Similr hnges in ICCs were found in the SGES (dibeti with shm GES) group (C). However, numerous ICCs were observed in the (dibeti group) + GES1 (GES prmeter 1) group (D), + GES2 group (E) nd + GES3 group (F). Sle brs = 100 μm. GES: Gstri eletril stimultion. α-sma IGF-1R DAPI Merge α-sma A B C D E F IGF-1R DAPI Merge Figure 7 Confol mirogrphs showing the distribution of insulin-like growth ftor 1 reeptor. The smooth musle ells were lbelled with α-sma (green), IGF-1R (red) nd DAPI (blue). IGF-1R expression ws high in the ontrol group (A). Wek IGF-1R signls were found in the group (B), nd similr hnges ppered in the SGES group (dibeti with shm GES) (C). Strong IGF-IR signls were ptured in the (dibeti group) + GES1 (GES prmeter 1) group (D), + GES2 group (E) nd + GES3 group (F). Sle brs = 100 μm. : nd GES prmeter 1. GES: Gstri eletril stimultion; IGF: Insulin-like growth ftor. [8,9] whih supports the findings of previous studies. Moreover, serum IGF-1 levels were lso deresed in the group. However, few studies hve exmined the effets of GES on IGF-1 nd IGF-1R levels or the involvement of IGF-1 signling in the indution of ICC remodeling by GES. In our study, the levels of both IGF-1 nd IGF-1R were signifintly inresed following GES tretment, whih mirrored the hnges we observed in M-SCF. Additionlly, GES2 (5.5 pm, 300 ms, 4 ma) ws more effetive in improving WJG the expression of IGF-1 nd IGF-1R. These results indite tht improvements in IGF-1 signling might be involved in the up-regultion of M-SCF expression nd tht it might lso ontribute to ICC remodeling in response to long-pulse GES in dibeti rts. [30] Xing et l reported tht the totl re under the urve for blood gluose in helthy dogs ws mrkedly deresed by long-pulse GES (10 pm, 300 ms, 8 ma). In the present study, GES did not signifintly ffet blood gluose levels. We speulte tht the 5361 June 21, 2016 Volume 22 Issue 23

10 Li H et l. Mehnisms by whih long-pulse GES ffets interstitil ells of Cjl differene between these effets might be ssoited with differenes in the prmeters, niml models nd physil sttes tht were used, nd tht the effet of GES on the blood gluose levels in dibeti models might therefore require further study. Yn et l [31] reported tht pulse-trin GES (0.3 ms, 3 ma, 20 Hz for 2 s on nd 3 s off) redued body weight in obese rts. Our results show tht long-pulse GES tretment mrkedly inresed body weight in dibeti rts. Therefore, the effets of GES on body weight might depend on the prmeters nd physil sttes tht re used. Brodly, our results suggest tht GES inresed the body weight but did not signifintly ffet blood gluose levels in STZ-indued dibeti rts. In summry, our results revel tht the expression levels of M-SCF, IGF-1 nd IGF-1R re signifintly lower nd tht the number of ICCs is mrkedly deresed in the gstri ntrum of dibeti rts. These hrteristis likely ontribute to delyed gstri emptying. Following long-pulse GES intervention, ICCs remodeled nd gstri emptying ws improved. The IGF-1 signling pthwy might prtiipte in this proess by enhning the expression of M-SCF on SMCs nd then proteting ICCs. Furthermore, the effets of GES2 (5.5 pm, 300 ms, 4 ma) on the expression of M-SCF nd IGF-1/IGF-1R ppered to be stronger thn those of GES1 nd GES2. Long-pulse GES involves low-frequeny/high-energy stimultion; its frequeny is slightly higher thn the intrinsi slow wve nd requires high mount of energy. This stimulus ould diretly tivte ICCs nd/or SMCs without involving holinergi nerves [21], nd the mgnitude of energy is probbly responsible for the effets of long-pulse GES. In our study, GES2 (5.5 pm, 300 ms, 4 ma) fforded higher energy thn did GES1 (5.5 pm, 100 ms, 4 ma) nd GES3 (5.5 pm, 550 ms, 2 ma), nd this might prtly explin why GES2 hd more signifint effet on gstri emptying nd the IGF-1 signling pthwy. Thus, this prmeter should be used in linil pplitions involving long-pulse GES. However, further studies into the mehnisms underlying these proesses re needed to ensure the sfety nd linil effiy of using longpulse GES s tretment for refrtory funtionl gstri disorders. COMMENTS Bkground Gstri eletril stimultion (GES) regultes gstri motility nd promotes the renovtion of interstitil ells of Cjl (ICCs). However, the mehnisms underlying this effet remin unler. Previous studies demonstrted tht ICCs re dmged nd the insulin-like growth ftor 1 (IGF-1) pthwy is downregulted in dibeti models. In this study, the uthors report tht pplying longpulse GES using three different prmeters promoted the regenertion of ICCs nd tht the IGF-1 signling pthwy is likely to be involved in this proess. Reserh frontiers Experiments hve shown tht ICCs re dmged in dibeti models. The -kit/scf pthwy is one of the most importnt regultors of ICCs. M-stem ell ftor (SCF) my ply more importnt role thn S-SCF in the differentition, survivl nd mintenne of the ICC phenotype. IGF-1 signling is one of the min ftors tht regulte the expression of M-SCF. Innovtions nd brekthroughs This study provides evidene showing tht long-pulse GES protets the ICCs in dibeti rts nd tht the IGF-1 signling pthwy is involved in this effet. The effets of one GES prmeter (5.5 pm, 300 ms, 4 ma) were stronger. Applitions One GES prmeter (5.5 pm, 300 ms, 4 ma) showed greter potentil for being used in linil pplition involving long-pulse GES, nd the IGF-1 signling pthwy my be possible therpeuti trget in gstropresis. Terminology ICCs, s the gstrointestinl pe-mking ells, ply n importnt physiologil role in oordinting gstri ontrtile tivity nd gstri motility. Long-pulse GES restores injured ICCs. SCF nd IGF-1 re regultory proteins tht protet ICCs. Peer-review An interesting pper! The pper by Li nd ollegues desribes the effet of different timing pulse GES (gstri eletril stimultion) on gstri emptying in rt dibeti model. In prtiulr, the uthors demonstrte tht long-term pulsing GES protets ICCs loted in the gstri ntrum nd this is ssoited with n improvement of gstri emptying dely during dibetes. Interestingly, the uthors lso suggest tht the IGF-1 signling pthwy my be involved. REFERENCES 1 Hirst GD, Edwrds FR. Eletril events underlying orgnized myogeni ontrtions of the guine pig stomh. J Physiol 2006; 576: [PMID: DOI: / jphysiol ] 2 Wng XY, Lmmers WJ, Berik P, Huizing JD. Lk of pylori interstitil ells of Cjl explins distint peristlti motor ptterns in stomh nd smll intestine. Am J Physiol Gstrointest Liver Physiol 2005; 289: G539-G549 [PMID: DOI: / jpgi ] 3 Torihshi S, Nishi K, Tokutomi Y, Nishi T, Wrd S, Snders KM. Blokde of kit signling indues trnsdifferentition of interstitil ells of jl to smooth musle phenotype. Gstroenterology 1999; 117: [PMID: ] 4 Tong W, Ji H, Zhng L, Li C, Ridolfi TJ, Liu B. Exogenous stem ell ftor improves interstitil ells of Cjl restortion fter blokde of -kit signling pthwy. Snd J Gstroenterol 2010; 45: [PMID: DOI: / ] 5 Forster J, Dmjnov I, Lin Z, Srosiek I, Wetzel P, MCllum RW. Absene of the interstitil ells of Cjl in ptients with gstropresis nd orreltion with linil findings. J Gstrointest Surg 2005; 9: [PMID: DOI: /j.gssur ] 6 Wng XY, Huizing JD, Dimond J, Liu LW. Loss of intrmusulr nd submusulr interstitil ells of Cjl nd ssoited enteri nerves is relted to deresed gstri emptying in streptozotoinindued dibetes. Neurogstroenterol Motil 2009; 21: 1095-e92 [PMID: DOI: /j x] 7 Ordög T, Tkym I, Cheung WK, Wrd SM, Snders KM. Remodeling of networks of interstitil ells of Cjl in murine model of dibeti gstropresis. Dibetes 2000; 49: [PMID: ] 8 Horváth VJ, Vittl H, Lörinz A, Chen H, Almeid-Pord G, Redelmn D, Ordög T. Redued stem ell ftor links smooth myopthy nd loss of interstitil ells of jl in murine dibeti gstropresis. Gstroenterology 2006; 130: [PMID: DOI: /j.gstro ] 9 Wng Y, Xu XY, Tng YR, Yng WW, Yun YF, Ning YJ, Yu YJ, WJG June 21, 2016 Volume 22 Issue 23

11 Li H et l. Mehnisms by whih long-pulse GES ffets interstitil ells of Cjl Lin L. Effet of endogenous insulin-like growth ftor nd stem ell ftor on dibeti oloni dysmotility. World J Gstroenterol 2013; 19: [PMID: DOI: /wjg.v19.i ] 10 D Addio F, L Ros S, Mestroni A, Jung P, Orsenigo E, Ben Nsr M, Tezz S, Bssi R, Finzi G, Mrndo A, Vergni A, Frego R, Albrello L, Andolfo A, Mnuguerr R, Vile E, Studher C, Corrdi D, Btlle E, Breult D, Sehi A, Folli F, Fiorin P. Cirulting IGF-I nd IGFBP3 Levels Humn Coloni Stem Cell Funtion nd Are Disrupted in Dibeti Enteropthy. Cell Stem Cell 2015; 17: [PMID: DOI: / j.stem ] 11 Li C, Liu S, Gun Y, Qin W, du F, Hou X. Long pulse gstri eletril stimultion indues regenertion of myenteri plexus synpti vesiles in dibeti rts. Neurogstroenterol Motil 2010; 22: , e108 [PMID: DOI: / j x] 12 Liu J, Qio X, Mii MA, Psrih PJ, Chen JD. Improvement of gstri motility with gstri eletril stimultion in STZ-indued dibeti rts. Digestion 2004; 70: [PMID: DOI: / ] 13 Du F, Wng L, Qin W, Liu S. Loss of enteri neurons ompnied by deresed expression of GDNF nd PI3K/Akt pthwy in dibeti rts. Neurogstroenterol Motil 2009; 21: 1229-e114 [PMID: DOI: /j x] 14 Chen Y, Xu J, Liu S, Hou X. Eletroupunture t ST36 inreses ontrtion of the gstri ntrum nd improves the SCF/-kit pthwy in dibeti rts. Am J Chin Med 2013; 41: [PMID: DOI: /s x ] 15 Xu X, Qin L, Chen JD. Anti-dysrhythmi effets of long-pulse gstri eletril stimultion in dogs. Digestion 2004; 69: [PMID: DOI: / ] 16 Xing J, Brody F, Rosen M, Chen JD, Soffer E. The effet of gstri eletril stimultion on nine gstri slow wves. Am J Physiol Gstrointest Liver Physiol 2003; 284: G956-G962 [PMID: DOI: /jpgi ] 17 Lei Y, Chen JD. Effets of dul pulse gstri eletril stimultion on gstri tone nd ompline in dogs. Dig Liver Dis 2009; 41: [PMID: DOI: /j.dld ] 18 Xu X, Brining DL, Chen JD. Effets of vsopressin nd long pulselow frequeny gstri eletril stimultion on gstri emptying, gstri nd intestinl myoeletril tivity nd symptoms in dogs. Neurogstroenterol Motil 2005; 17: [PMID: DOI: /j x] 19 Chen JD, Qin L, Ouyng H, Yin J. Gstri eletril stimultion with short pulses redues vomiting but not dysrhythmis in dogs. Gstroenterology 2003; 124: [PMID: DOI: /gst ] 20 Xing J, Brody F, Brodsky J, Rosen M, Lrive B, Ponsky J, Soffer E. Gstri eletril-stimultion effets on nine gstri emptying, food intke, nd body weight. Obes Res 2003; 11: [PMID: DOI: /oby ] 21 Bortolotti M. Gstri eletril stimultion for gstropresis: gol gretly pursued, but not yet ttined. World J Gstroenterol 2011; 17: [PMID: DOI: /wjg.v17.i3.273] 22 Song GQ, Lei Y, Xu X, Chen JD. Gstri eletril stimultion with long pulses in humns nd nimls: n dt obtined in nimls be replited in humns? Neuromodultion 2010; 13: [PMID: DOI: /j x] 23 Bellhsène BE, Lind CD, Shirmer BD, Updike OL, MCllum RW. Aelertion of gstri emptying with eletril stimultion in nine model of gstropresis. Am J Physiol 1992; 262: G826-G834 [PMID: ] 24 MCllum RW, Chen JD, Lin Z, Shirmer BD, Willims RD, Ross RA. Gstri ping improves emptying nd symptoms in ptients with gstropresis. Gstroenterology 1998; 114: [PMID: ] 25 Vittl H, Frrugi G, Gomez G, Psrih PJ. Mehnisms of disese: the pthologil bsis of gstropresis-- review of experimentl nd linil studies. Nt Clin Prt Gstroenterol Heptol 2007; 4: [PMID: DOI: /npgsthep0838] 26 Mitsui R, Komuro T. Distribution nd ultrstruture of interstitil ells of Cjl in the gstri ntrum of wild-type nd Ws/Ws rts. Ant Embryol (Berl) 2003; 206: [PMID: DOI: /s ] 27 Rih A, Miller SM, Gibbons SJ, Mlysz J, Szurszewski JH, Frrugi G. Lol presenttion of Steel ftor inreses expression of -kit immunoretive interstitil ells of Cjl in ulture. Am J Physiol Gstrointest Liver Physiol 2003; 284: G313-G320 [PMID: DOI: /jpgi ] 28 Wrd SM, Ordög T, Byguinov JR, Horowitz B, Epperson A, Shen L, Westphl H, Snders KM. Development of interstitil ells of Cjl nd pemking in mie lking enteri nerves. Gstroenterology 1999; 117: [PMID: ] 29 Horváth VJ, Vittl H, Ordög T. Redued insulin nd IGF-I signling, not hyperglyemi, underlies the dibetes-ssoited depletion of interstitil ells of Cjl in the murine stomh. Dibetes 2005; 54: [PMID: ] 30 Xing JH, Lei Y, Anh HR, Hrty RF, Chen JD. Effet of ute gstri eletril stimultion on the systemi relese of hormones nd plsm gluose in dogs. Dig Dis Si 2007; 52: [PMID: DOI: /s x] 31 Yn Y, Xing XL, Qin W, Xu JY, Hou XH. Chnges of neuronl tivities fter gut eletril stimultion with different prmeters nd lotions in lterl hypothlmus re of obese rts. J Huzhong Univ Si Tehnolog Med Si 2014; 34: [PMID: DOI: /s z] P- Reviewer: Blir PJ, Fiorin P, Kito Y S- Editor: Yu J L- Editor: Wng TQ E- Editor: Wng CH WJG June 21, 2016 Volume 22 Issue 23

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