Therapeutic Effects of Berberine Hydrochloride on Stress-Induced Diarrhea-Predominant Irritable Bowel Syndrome Rats by Inhibiting Neurotransmission in Colonic Smooth Muscle

Abstract

The etiology of diarrhea-predominant irritable bowel syndrome (IBS-D) is complicated and closely related to neurotransmission in the gastrointestinal (GI) tract. Developing new strategies for treating this disease is a major challenge for IBS-D research. Berberine hydrochloride (BBH), the derivative of berberine, is a herbal constituent used to treat IBS. Previous studies have shown that BBH has potential anti-inflammatory, antibacterial, analgesic, and antidiarrheal effects and a wide range of biological activities, especially in regulating the release of some neurotransmitters. A modified IBS-D rat model induced by chronic restraint stress was used in all experiments to study the effects of BBH on the GI tract. This study measured the abdominal withdrawal reflex (AWR) response to graded colorectal distention (CRD; 20, 40, 60, and 80 mmHg) and observed the fecal areas of stress-induced IBS-D model. Experiments were conducted using organ bath techniques, which were performed in vitro using strips of colonic longitudinal smooth muscle. Inhibitory and excitatory neurotransmitter agents were added to each organ bath to observe contractile responses on the strips and the treatment effect exerted by BBH. The IBS-D rat model was successfully induced by chronic restraint stress, which resulted in an increased defecation frequency and visceral hypersensitivity similar to that of humans. BBH could reduce 4-h fecal areas and AWR response to CRD in IBS-D. The stress-induced IBS-D model showed upregulated colonic mRNA expression levels of 5-hydroxytryptamine-3A receptor and downregulated expression levels of neuronal nitric oxide synthase. Meanwhile, BBH could reverse this outcome. The responses of substances that regulate the contraction induced by related neurotransmission in the longitudinal smooth muscle of IBS-D colon (including the agonist of acetylcholine, carbachol; NOS inhibitor, L-NAME; and P2Y₁ receptor antagonist, MRS2500) can be inhibited by BBH. In summary, BBH promotes defecation frequency and visceral hypersensitivity in IBS-D and exerts inhibitory effects on contractile responses in colonic longitudinal smooth muscle. Thus, BBH may represent a new therapeutic approach for treating IBS-D.

Keywords: berberine hydrochloride; colonic longitudinal smooth muscles; diarrhea-predominant irritable bowel syndrome; inhibitory effects; neurotransmission.

FIGURE 1

Structure of BBH.

FIGURE 2

Graph of the excrement before and after ImageJ analysis.

FIGURE 3

Establishment of the IBS-D rat model. (A,B) Measurement results of fecal areas and AWR scores at 40 and 60 mmHg on days 0, 7, 14, 21, and 28. (C) Box and whisker plots of the distribution and mean expression levels of 5-HT_3AR and nNOS in the colonic tissues of the control and IBS-D groups. Data are represented as mean ± SEM. n = 6–12 rats per group. *denotes p < 0.05 compared with the control group.

FIGURE 4

BBH ameliorated symptoms in IBS-D rats. Box and whisker plots of the distribution and mean of fecal areas, AWR scores, and the expression levels of 5-HT_3AR and nNOS of the control group, model groups, and four treatment groups on days 21 and 28. The AWR scores were measured in response to graded CRD (40 and 60 mmHg). n = 6–12 rats per group. ^*denotes p < 0.05 compared with the control group. ^#denotes p < 0.05 compared with the model group.

FIGURE 5

BBH inhibited spontaneous and EFS-induced contractions of TTX-treated isolated colonic longitudinal smooth muscle. (A,B) Mechanical recording and linear regression curve of the cumulative log concentration–response of the effect of BBH (1.5625–200 μmol L⁻¹) on the spontaneous contractions of the isolated colonic longitudinal smooth muscle. The EC₅₀ of BBH was 25.17 μmol/L. DMSO had no effect. *denotes p < 0.01 compared with the vehicle DMSO group. BBH: n = 10, DMSO: n = 7. (C–J) Mechanical recording (C–H) and histogram (I–J) of the effect of TTX and BBH + TTX treatments on the EFS-induced contraction response of colonic longitudinal smooth muscle in the control and IBS-D groups. (C): EFS-induced control group (n = 16). (D): EFS-induced IBS-D group (n = 22). (E): TTX-treated control group (n = 12). (F): TTX-treated model group (n = 14). (G): BBH + TTX-treated control group (n = 11). (H): BBH + TTX-treated IBS-D group (n = 8). (I): BBH inhibited EFS-induced contractions of TTX-treated colonic tissues in the control group. # denotes p < 0.05 compared with the EFS-induced control group & denotes p < 0.05 compared with the TTX-treated control group. (J): BBH inhibited the EFS-induced contractions of TTX-treated colonic tissues in the IBS-D group. $ denotes p > 0.05 compared with the EFS-induced IBS-D group. Error bars represent mean ± SEM.

FIGURE 6

BBH inhibited the cholinergic contractile responses of isolated colonic longitudinal smooth muscles. Mechanical recording (A–D) and cumulative log concentration–response curve (E–G) of the effect of CCh and BBH + CCh treatments on the contractions of isolated colonic longitudinal smooth muscles in the control and IBS-D groups. (A): CCh-induced control group (n = 24). (B): BBH + CCh-induced control group (n = 18). (C): CCh-induced IBS-D group (n = 8). (D): BBH + CCh-induced IBS-D group (n = 15). (E): Cholinergic contractile responses in the control and IBS-D groups. The EC₅₀ obtained in the IBS-D group was 0.4906 μmol/L, whereas that in the control group was 2.527 μmol/L * denotes p < 0.05 compared with the CCh-induced control group. (F): BBH inhibited the cholinergic contractile responses in the control group. * denotes p < 0.05 compared with the CCh-induced control group. (G): BBH inhibited the cholinergic contractile responses in the IBS-D group. # denotes p < 0.05 compared with the CCh-induced IBS-D group. Error bars represent mean ± SEM.

FIGURE 7

BBH inhibited the EFS-induced nitrergic contractile responses of isolated colonic longitudinal smooth muscles. Mechanical recording (A–F) and line chart (G–J) of the effect of L-NAME and BBH + L-NAME treatments on the EFS-induced contraction response of colonic longitudinal smooth muscles in the control and IBS-D groups. (A): EFS-induced control group (n = 16) (B): EFS-induced IBS-D group (n = 22) (C): L-NAME-induced control group (n = 24). (D): L-NAME-induced IBS-D group (n = 22). (E): BBH + L-NAME-induced control group (n = 16). (F): BBH + L-NAME-induced IBS-D group (n = 14). (G): EFS-induced contractile responses in the control and IBS-D groups. * denotes p < 0.05 compared with the EFS-induced control group. (H): EFS-induced nitrergic contractile responses in the control and IBS-D groups. * denotes p < 0.05 compared with the EFS-induced control group; # denotes p < 0.05 compared with the EFS-induced IBS-D group. (I): BBH inhibited the EFS-induced nitrergic contractile responses in the control groups & denotes p < 0.05 compared with the L-NAME-induced control group. (J): BBH inhibited the EFS-induced nitrergic contractile responses in the IBS-D groups. $ denotes p < 0.05 compared with the L-NAME-induced IBS-D group. Error bars represent mean ± SEM.

FIGURE 8

BBH inhibited the EFS-induced contractile responses of isolated colonic longitudinal smooth muscles treated with P2Y₁ receptor antagonist. Mechanical recording (A, B, and E–J), histogram (C,D), and line chart (K,L) of the inhibitory effect of BBH on the tension of the α,β-MeATP-treated and MRS2500-treated isolated colonic longitudinal smooth muscles in the control and IBS-D groups. (A): α,β-MeATP-treated (n = 24) and MRS2500-treated (n = 24) control groups. (B): α,β-MeATP-treated (n = 24) and MRS2500-treated (n = 24) IBS-D groups. (C): *denotes p < 0.05 compared with the α,β-MeATP-treated group. (D): # denotes p < 0.05 compared with the MRS2500-treated control group. (E) EFS-induced control group (n = 18). (F): IBS-D group (n = 14). (G): EFS + MRS2500-treated control group (n = 24). (H): EFS + MRS2500-treated IBS-D group (n = 29). (I): BBH + MRS2500-treated and EFS-induced control group (n = 25). (J): BBH + MRS2500-treated and EFS-induced IBS-D group (n = 14). (K): Inhibitory effect of BBH on the MRS2500-treated control group. (L): Inhibitory effect of BBH on the MRS2500-treated IBS-D group & denotes p < 0.05 compared with the EFS-induced IBS-D group. $ denotes p < 0.05 compared with the EFS + MRS2500-induced IBS-D group. Error bars represent mean ± SEM.