Effect Of The Ethanol Extract Of Andrographis Paniculata On The Diarrhea In Mice And The Isolated Jejunumsmooth Muscle In Rabbits And Its Mechanism

Objective:To explore the antidiarrhea activity of the ethanol extract ofAndrographis Paniculata(Burm.F.)Nees(AP ext)and its effect on isolated jejunum smooth muscle on rabbits.Methods:The chemical constituents of AP ext were tested by HPLC.In vivo:Fifty male Kunming mice were randomly divided into 5 groups:Model group,loperamide group(4 mg/kg)and AP ext groups(250/500/1000 mg/kg).Diarrhea models were established by intragastric administration of castor oil orsenna leavesor sodium sulfate,respectively.Evacuation Index(EI)was to evaluate the antidiarrhea activity of AP ext.Fifty male Kunming mice were randomly divided into 5 groups:Model group,loperamide group(4 mg/kg)and AP ext groups(250/500/1000 mg/kg).Diarrhea model was established by intragastric administration of castor oil,and small intestine propulsion rate was determined by ink propulsion test.Seventy-two Kunming mice(male and female half)were randomly divided into 9 groups:Model group,negative control group,loperamide group(4 mg/kg),AP ext(250/500/1000 mg/kg)test groups and andrographolide(75/150/300mg/kg)test groups.Diarrhea model was established by intragastric administrationof senna leaves for 5 days,drug treatment for 2 days,and the mice was sacrificed after 12 hours of fasting.The protein expression level of the transient receptor potential channel-ankyrin 1(TRPA1),5-hydroxytryptamine receptor 3(5-HT3R),aquaporin 4(AQP4)and Na+/H+ exchanger 3(NHE3)in colon was tested by immunohistochemistry(IHC).The mRNA level of TRPA1,5-HT3R,AQP4 and NHE3 in colon was tested by quantitative reverse transcription polymerase chain reaction(qRT-PCR).In vitro:The effect of AP ext(0.01-10 mg/mL),andrographolide(0.3-300 μM)and apigenin(0.3-300 μM)on spontaneous contraction of rabbit jejunum smooth muscle was observed.Verapamil(0.01-10μM)was used as the positive control group.Jejunum was pretreated with ACh(10-5 M)to observe the inhibitory effect of AP ext(0.03-10 mg/mL)on ACh-induced contraction.Atropine(0.003-1 μM)was used as positive control group.Jejunum was pretreated with KCl(25 mM)or KCl(60 mM)to observe the inhibitory effect of AP ext(0.01-10 mg/mL).Cromakalim(0.001-10 μM)and verapamil(0.001-10 μM)were used as positive control group.Jejunum was pre-incubated by Ca2+-free high K+Tyrode solution with EDTAfor 30min to eliminate Ca2+ in the tissues,and then the jejunum was pre-incubated by Ca2+-free high K+Tyrode solution without EDTAfor 15min.After AP ext(0.3,1 mg/mL),CaCl2 was added cumulatively(3 10-5-3 10-2 M),and the concentration-response curves of CaCl2 were drawn.Verapamil(0.03,0.1 μM)was set as positive control group.Results:HPLC analysis showed that AP ext contained andrographolide and apigenin.In vivo:In senna leaves diarrhea model,compared with the model group,AP ext of 500 and 1000 mg/kg reduced the EI significantly(p<0.01;p<0.001),loperamide control group(4 mg/kg)reduced the EI of mice significantly(p<0.001);In castor oil diarrhea model,compared with the model group,AP ext of 250,500 and 1000 mg/kg reduced the EI(p<0.05;p<0.01;p<0.001),loperamide control group(4 mg/kg)reduced the EI of mice significantly(p<0.001);In sodium sulfate diarrhea model,compared with model group,AP ext of 250,500 and 1000 mg/kg reduced EI(p<0.05),loperamide control group(0.4 mg/kg)reduced the El of mice significantly(p<0.01).In gastrointestinal motility test,compared with model group,AP ext of 250 mg/kg reduced the intestinal propulsion rate of mice(p<0.05).AP ext of 500/1000 mg/kg and loperamide group(4 mg/kg)could reduce the small intestine propulsion rate of mice significantly(p<0.001).IHC results showed that compared with the model group,the positive expression of TRPA1 and 5-HT3R in the proximal colon tissues was significantly decreased after treatment with AP ext and andrographolide in the high dose group and loperamide group(p<0.001,p<0.01),the positive expression of TRPA1 and 5-HT3R was decreased in the AP ext with the medium dose group(p<0.05);Compared with the model group,the positive expressions of AQP4 and NHE3 in the proximal colon tissues were significantly increased after treatment with AP ext in the high dose and loperamide group(p<0.01),the positive expression of AQP4 and NHE3 was decreased in the andrographolide with the high dose group(p<0.05).qRT-PCR results showed that compared with model group,mRNA expression of TRPA1 and 5-HT3R in proximal colon tissues was significantly decreased after AP ext and andrographolide in the high dose and loperamide treatment(p<0.01,p<0.001);Compared with the model group,the mRNA expression of AQP4 and NHE3 in the proximal colon tissues was significantly increased after AP ext and andrographolide in the high dose and loperamide treatment(p<0.01).In vitro:Andrographolide(0.3-300 μM),apigenin(0.3-300 μM),AP ext(0.01-10 mg/mL)and verapamil(0.01-10 μM)could inhibit spontaneous contraction of jejunum smooth muscle in rabbits.The EC50 values of AP ext and verapamil were 3.72 mg/mL(2.93-4.58 mg/mL,95%CI,n=6)and 2.76 μM(2.05-3.24 μM,95%CI,n=6),respectively.Both AP ext(0.03-10 mg/mL)and Verapamil(0.003-1 μM)inhibited ACh(10-5 M)-induced contraction,with EC50 values of 3.46 mg/mL(3.13-4.38 mg/mL,95%CI,n=6)and 0.22 μM(0.19-0.28 μM,95%CI,n=6);Both cromakalim(0.001-10 μM)and verapamil(0.001-10μM)inhibited KCl(25 mM)-induced contraction with EC50 values of 0.37 mg/mL(0.28-0.51,95%CI,n=6),1.18 μM(0.91-1.35 μM,95%CI,n=6)and 0.17 μM(0.11-0.24 μM,95%CI,n=6);Both AP ext(0.03-10 mg/mL)and verapamil(0.001-10 μM)inhibited KCl(60 mM)-induced contraction with EC50 values of 2.45 mg/mL(2.13-2.87 mg/mL,95%CI,n=6)and 0.87μM(0.74-0.92 μM,95%CI,n=6).Cromakalim(0.01-10 μM)had no inhibitory effect on the contraction of jejunum smooth muscle induced by KCl(60 mM).In the Tyrode solution group,the maximum contraction rate caused by CaCl2 of 30 mM was set as 100%.Pretreatment of 0.03 and 0.1μMverapamil reduced the maximum contraction rate to 47.91%±4.50%and 13.24%±3.30%.Similar to verapamil,0.03 and 1 mg/mL AP ext reduced the maximum contraction rate to 62.02%±4.68%and 18.65%±4.83%.Conclusion:In vivo experiments showed that AP ext has antidiarrhea activity,the mechanism may be related to the decrease of TRPA1 and 5-HT3R to inhibit gastrointestinal motility and the increase of NHE3 and AQP4 to increase intestinal absorption of water and electrolyte;In vitro experiments showed that AP ext inhibited the contraction of isolated jejunal smooth muscle of rabbits,and its mechanism might be related to blocking M receptors and L-type voltage-gated Ca2+ channels.