| Background: Evodiamine(EVO)is a major alkaloid compound extracted from the dry unripened fruit Evodiae fructus(Evodia rutaecarpa Benth.,Rutaceae).As a natural indole alkaloid,EVO has many biological activities.It has been recognized by many scholars for its anti-tumor activity and gastric mucosal protection.At the same time,Evodiamine also shows potential value in regulating gastrointestinal motility and visceral sensitivity,but the current evidence is not yet enough.The three major factors involved in intestinal motility regulation are mainly Enteric nervous system,Cajal cells(ICC)and smooth muscle.They are also the three major targets of gastrointestinal motility drugs.L-type calcium channels are expressed both in intestinal smooth muscle and submucosa layers,which are involved in the regulation of smooth muscle contraction and cell excitability.As an inhibitory gas molecule,NO is involved in regulating gastrointestinal motility.EVO is highly correlated with NO production in cardiovascular system research.Other studies have reported that evodiamine can regulate the release of gastrointestinal hormones and brain-derived neurotrophic factors,and participate in pain regulation by activating capsaicin receptors TRPV1.Irritable bowel syndrome(IBS)is one of the most common functional gastrointestinal diseases,it is mainly manifested by abdominal discomfort and/or pain,chronic intermittent defecation habits and changes in stool characteristics,and lack of abnormal morphological and biochemical changes.At present,it is widely accepted that the disorder of gastrointestinal motility and visceral hypersensitivity are closely related to the occurrence and development of this disease.The purpose of this study was to investigate the role of evodiamine in regulating gastrointestinal motility and visceral sensitivity,and to provide theoretical basis for the treatment of irritable bowel syndrome and functional gastrointestinal diseases.Evodiamine has been recommended for gastrointestinal disorders such as abdominal pain,acid deficiency,nausea and diarrhea.Part ? Effect of Evodiamine on Gastrointestinal Motility in Rats Subjected to Chronic Water Avoidance Stress and the Potential Mechanisms Objective: To investigate the effect of evodiamine on gastrointestinal motility in rats subjected to chronic water avoidance stress and its related mechanismsMethods: Healthy male SD rats were selected as subjects of this study,the animal model was established by chronic water avoidance stress,To determine the effect of EVO on gastrointestinal propulsion in rats,we calculated the number of feces in 24 hours and the time of the first black stool discharged after intragastric administration of Indian ink;The colonic contractile activity was studied in an organ bath system,The serum CCK-8 level was detected using an enzyme immunoassay kit;The protein level of NO synthase in rat colon tissue was detected by Western blotting;The whole-cell patch clamp technique was used to observe the effect of EVO on the current density of L-type calcium channels and BKCa channels and the effect on steady-activation /inactivation of L-type calcium channels.Results: The gastrointestinal motility was significantly increased in rats subjected to water avoidance stress;The mean contractile amplitudes of both the LM and CM strips were significantly higher for the WAS rats than for the SWAS rats(LM: 1.22±0.08 vs 0.69±0.03,P<0.05;CM: 0.80±0.06 vs 0.52±0.04,P<0.05);EVO significantly inhibited the contractile activity of colonic smooth muscle strips,for the LM strips,the mean contractile amplitude before adding EVO was 1.11±0.12 g,whereas the amplitude in the presence of EVO(10,20,and 40 ?M)dropped to 0.77±0.06 g,0.45±0.07 g,and 0.28±0.06 g,respectively(P<0.05 vs.the control).Similarly,before the addition of EVO,the mean amplitude of the CM strips was 0.74±0.09 g and after adding EVO(5,10,and 20 ?M),the amplitude decreased to 0.55±0.03 g,0.26±0.02 g,and 0.13±0.007 g respectively,both 10?M and 20?M EVO incubation can significantly inhibit the contraction of CM strips(P<0.05 vs.the control);this effect could not be blocked by TTX;after removing mucosal and sub-mucosal,the inhibitory effect of EVO still exist and can't be blocked by TTX;EVO elevated the serum CCK-8 level in the WAS rats in a dose-dependent manner;Exogenous CCK-8 significantly inhibited the contractile activity of the colonic muscle strips;this effect was not blocked by TTX but was completely blocked by devazepide;Both EVO and CCK-8 inhibited gastrointestinal transit,and the effect of EVO could be partially blocked by devazepide;the protein level of n NOS in colon tissue of EVO administrated rat was significantly increased.Conclusion: The gastrointestinal propulsion and colonic spontaneous contraction were significantly increased in rats subjected to chronic water avoidance stress.EVO can effectively inhibit gastrointestinal transmission and colonic spontaneous contraction in rats and these effects may be achieved by promoting the production of NO and CCK-8,and directly attenuate the current of L-type calcium channels in smooth muscle cells.Part ? Effect of Evodiamine on L-type calcium channels and BKCa channels in smooth muscle cells of rat colonObjectives: To investigate the effects of evodiamine on L-type calcium channel and large conductance calcium-dependent potassium channel in rat smooth muscle cellsMethods: Isolated smooth muscle strips without mucosa and submucosa were prepared,The effects of EVO on contractile activity of proximal colon smooth muscle in rats were detected by RM6240 multi-channel physiological signal acquisition system.Collagenase type II digestion method was used to isolate colonic smooth muscle cells.whole-cell patch clamp technique was used to detect the effect of evodiamine on the current density and steady-state activation and inactivation of L-type calcium channel under voltage clamp mode.The same method was used to detect the effect of evodiamine on the current of large-conductance calciumdependent potassium channel.Results: After removing the mucosa and submucosa,the inhibitory effect of EVO on the spontaneous contraction of rat colonic smooth muscle strips still exist.Before incubation with EVO,the mean contractile amplitude of LM and CM strips were(0.82± 0.12)g,(0.47 ± 0.02)g,after the administration of EVO the mean contractile amplitude of LM and CM strips dropped to(0.49± 0.14)g,(0.29±0.05)g respectively,(P<0.05 vs.the control),and the effect could not be blocked by TTX.Whole-cell patch clamp technique was used to detect the effect of EVO on the current of L-type calcium channel.The results showed that the peak current appeared at 0 m V.Before incubation with EVO,the average peak current density was(5.17 +0.35)p A/p F.After incubation with different concentrations of EVO(5?M,10?M,20?M),the peak current density decreased to(3.10 +0.16)p A/p F,(-2.98 +0.26)p A/p F,(1.39 +0.22)p A/p F,respectively,(P<0.05 vs control).When incubated with the same dose of DMSO(1,2,4 ul),we have not detected any significant change in the current curve and the peak current density.After elution,the current partially recovered.From the I-V curve we can see that EVO can inhibit the current of L-type calcium channel under any impulse stimulation,but EVO does not change the shape of I-V curve,and so does the same dose of DMSO.EVO had no significant effect on the steady-state activation of L-type calcium channels.Before and after incubation,the V1/2 was(18.78 ±2.42)m V,(-19.73 ±2.30 m V),respectively,(P > 0.05,n = 5).the ? value changed from 3.94 ±2.53 to 3.78 ±2.71,(P > 0.05,n = 5).EVO had no significant effect on the steady-state inactivation of L-type calcium channels.The V1/2 value before and after the incubation with EVO(10?M)were(-30.96 ±-0.48)m V vs(-32.24 ±-0.48)m V),(P > 0.05,n = 5),the ? value changed from(5.56±0.43)to(5.19±0.41),(P>0.05,n=5).EVO(5?M,10?M)can dose-dependently inhibit the current density of large conductance calcium-dependent potassium channels under each test pulse,the peak current appears at + 60 m V,but do not change the shape of I-V curve.The average peak current density before incubation with EVO was(17.93(+1.49)p A/p F,EVO(5?M,10?M)decreased the current density to(12.13 ±1.23)p A/p F,(5.73 ±1.56)p A/p F,respectively,(P<0.05,n=5).conclusion:The inhibitory effect of EVO on spontaneous contraction of colonic strips is not completely mucosal dependent.EVO reversibly inhibit the current of L-type calcium channels without changing the steady-activation and steadyinactivation in colonic smooth muscle cells.EVO dose dependently inhibit the current density of large conductance calcium-dependent potassium channel.Part ? Effect of Evodiamine on Visceral Sensitivity in Rats Subjected to Chronic Water Avoidance Stress and Its Molecular MechanismsObjectives:To investigate the effect of evodiamine on visceral sensitivity in rats subjected to chronic water avoidance stress and its potential Molecular mechanisms.Methods: In this study,the healthy adult male SD rats were exposed to daily 1-h water avoidance stress(WAS)or sham WAS for 10 consecutive days to establish the animal model.The visceral sensitivity to CRD was detected by power-lab,western blot was performed to assess the expression of TRPV1,p-TRPV1 and CGRP,Immunohistochemistry was performed to assess the SP in the colon tissue.Results: Repeated WAS increases the visceral sensitivity of rats,the AUCs of WAS rats were significantly increased compared with that in SWAS group at the extended stimulation of 1.0ml water(162.08±27.95 versus 360.4732±60.78 ?V*s,P<0.05).we have not detected any significant difference between the WAS and SWAS group at the extended stimulation of 0.6ml and 1.4ml water.Treatment with 0.67mg/kg EVO for 7 consecutive days can significantly improve the visceral hypersensitivity of rats induced by chronic water avoidance stress.At the extended stimulation of 0.6ml water,the average AUC of WAS,DMSO,EVO(L),EVO(M)and EVO(H)group were as follows:(124.31±6.90)?V *s,(157.58±28.05)?V*s,(216.01±18.44)?V*s,(186.05±32.78)?V*s,(121.64±29.09)?V*s,there was not any significant difference between them(P > 0.05,n=5).At the extended stimulation of 1.0ml water,the average AUC of the above groups were as follows:(366.00±49.93)?V*s,(306.27±18.19)?V*s,(294.44±22.36)?V*s,(215.59±31.24)?V*s,(193.07±30.29)?V*s,there were significant differences between EVO(H)group and WAS,DMSO group,(P<0.05,n=5);there was no significant difference between the other groups(P > 0.05,n=5);At the extended stimulation of 1.4ml water,the average AUC of the above groups were as follows:(469.30±24.09)?V*s,(365.53±31.81)?V*s,(433.80±29.08)?V*s,(434.14±68.92)?V*s,(351.60±27.23)?V*s;(P>0.05,n=5).The response of colonic longitudinal muscle strips to Ach stimulation was significantly lower in EVO(H)group than that in DMSO group,but this phenomenon was not detected in circular muscle strips,and there was no significant difference among the other groups both in longitudinal and circular muscle strips.WAS can significantly increase the protein level of TRPV1 but not p-TRPV1 in the colon tissue,and the protein level of TRPV1 and p-TRPV1 in EVO(M)and EVO(H)were significantly decreased compared with that in DMSOgroup;There was no significant difference in the expression of CGRP and SP between EVO administration group and the controls.Conclusion: The visceral sensitivity was significantly increased in rats subjected to chronic water avoidance stress.EVO(6mg/kg)could effectively reverse the visceral hypersensitivity induced by chronic water avoidance stress in rats.and this effect was not related to SP and CGRP,but probably through desensitization of TRPV1 receptor,however,it is not excluded that EVO can directly down-regulate TRPV1 expression in colon tissue. |
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