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. 2016 May 26:7:134.
doi: 10.3389/fphar.2016.00134. eCollection 2016.

Salvianolic Acid B Restored Impaired Barrier Function via Downregulation of MLCK by microRNA-1 in Rat Colitis Model

Yongjian Xiong  1 , Jingyu Wang  2 , Hongwei Chu  3 , Dapeng Chen  2 , Huishu Guo  1
Affiliations

Salvianolic Acid B Restored Impaired Barrier Function via Downregulation of MLCK by microRNA-1 in Rat Colitis Model

Yongjian Xiong et al. Front Pharmacol. .

Abstract

Salvianolic acid B (Sal B) is isolated from the traditional Chinese medical herb Salvia miltiorrhiza and is reported to have a wide range of therapeutic benefits. The aim of this study was to investigate the effects of Sal B on epithelial barrier dysfunction in rat colitis and to uncover related mechanisms. Rat colitis model was established by intracolonic administration of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS). The intestinal barrier function was evaluated by measuring the serum recovery of fluorescein isothiocyanate-4 kD dextran in vivo and transepithelial electrical resistance in vitro respectively. The protein expression related to intestinal barrier function was studied using western blotting. The effects of Sal B on inflammatory responses, oxidative damage and colitis recurrence were also studied in this study. The intestinal barrier dysfunction in colitis was reversed by Sal B, accompanied with the decrease of tight junction proteins, and the effect could be blocked by microRNA-1(miR-1) inhibition. The inflammatory responses, oxidative damage and colitis recurrence were also decreased by Sal B. The colitis symptoms and recurrences were ameliorated by Sal B, and restoration of impaired barrier function via downregulation of MLCK by miR-1 maybe involved in this effect. This study provides some novel insights into both of the pathological mechanisms and treatment alternatives of inflammatory bowel disease.

Keywords: barrier function; inflammatory bowel disease; microRNA-1; myosin light chain kinase; salvianolic acid B.

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Figures

Figure 1
Figure 1
Experimental flowchart for the present study.
Figure 2
Figure 2
Salvianolic acid B (Sal B) induced modulations on oxidative stress and epithelial barrier function in normal rats. After gavage administration of Sal B once a day for seven consecutive days, the contents of (A) NADPH oxidase 4 (NOX4), (B) inducible nitric oxide synthase (iNOS), (C) malondialdehyde (MDA), (D) glutathione (GSH), (E) superoxide dismutases (SOD), and (F) long myosin light chain kinase (MLCK) from colon tissue in normal control (NC) rats were observed using ELISA methods; (G) the effects of Sal B on the serum recovery of FD-4 was also observed. Data are expressed as the mean ± SD. **P < 0.01, compared with the NC group (n = 6). Data in the NC group is set to 100%; other data are the relative values compared with those in the NC group. Sal B(L), 20 mg/kg Sal B; Sal B(H), 80 mg/kg Sal B.
Figure 3
Figure 3
Salvianolic acid B (Sal B) ameliorated colitis symptoms. (A) Hematoxylin and eosin (HE)- staining analysis of rat colonic tissue. Effects of Sal B on (B) body weight, (C) food intake, (D) macroscopically visible damage, and (E) colon weight-to-length ratio in colitis rats. Data are expressed as the mean ± SD. **P < 0.01 compared with the sham group (n = 7); ##P < 0.01 compared with the TNBS control group (n = 7). SASP, sulfasalazine; Sal B(L), 20 mg/kg Sal B; Sal B(H), 80 mg/kg Sal B.
Figure 4
Figure 4
Salvianolic acid B (Sal B) reduced neutrophil infiltration and cytokine profiles. ELISA results of Sal B-induced effects on proinflammatory cytokine (A) TNF-α, (B) IL-6, (C) IL-1β, and (D) myeloperoxidase (MPO) from rat colon tissue. Data are expressed as the mean ± SD; **P < 0.01 compared with the sham group; ##P < 0.01 compared with the TNBS control group (n = 7). SASP, sulfasalazine; Sal B(L), 20 mg/kg Sal B; Sal B(H), 80 mg/kg Sal B.
Figure 5
Figure 5
Salvianolic acid B (Sal B) restored barrier function through decreasing intestinal permeability. (A) Effects of Sal B on the serum recovery of FD-4. Effects of Sal B on tight junction protein expression of claudin-2 and zona occludens ZO-1 from colon tissue using western blotting (B) and immunofluorescence technique (C). Data are expressed as the mean ± SD; **P < 0.01 compared with the sham group; ##P < 0.01 compared with the TNBS control group (n = 7). Data in the sham group is set to 100%; other data are the relative values compared with those in the sham group. SASP, sulfasalazine; Sal B(L), 20 mg/kg Sal B; Sal B(H), 80 mg/kg Sal B.
Figure 6
Figure 6
Salvianolic acid B (Sal B) reversed the altered microRNA-1(miR-1) and MLCK expression. Effects of Sal B on the expression of (A) miR-1 and (B) MLCK in rat colitis model was measured. Data are expressed as the mean ± SD; **P < 0.01 compared with the sham group; ##P < 0.01 compared with the TNBS control group (n = 7). Data in the sham group is set to 100%; other data are the relative values compared with those in the sham group. SASP, sulfasalazine; Sal B(L), 20 mg/kg Sal B; Sal B(H), 80 mg/kg Sal B.
Figure 7
Figure 7
MicroRNA-1(miR-1) mediated Sal B-induced restoration effects by targeting of MLCK. (A) Sal B abrogates the increase in MLCK following miR-1 repressed. (B–C) Sal B abrogates the increase in MLCK following miR-1 repressed. The data are presented as the mean ± SD. (n = 3). **P < 0.01 vs. the antago-miR control group, ##P < 0.01 vs. miR-1 or MLCK before Sal B treatment respectively.
Figure 8
Figure 8
Sal B-induced restoration effects was abolished by microRNA-1(miR-1) inhibition in vitro. (A) Sal B-induced inhibition on tight junction proteins claudin-2 and zona occludens (ZO-1) contents and (B) stimulation on transepithelial electrical resistance (TER) levels were abolished in the present of miR-1 inhibition. The data are presented as the mean ± SD. (n = 3). ##P < 0.01 vs. tight junction proteins or TER levels before Sal B treatment respectively.
Figure 9
Figure 9
Salvianolic acid B (Sal B) increased the antioxidant capacity. Effects of Sal B on protein expression of (A) NADPH oxidase 4 (NOX4), (B) inducible nitric oxide synthase (iNOS), (C) malondialdehyde (MDA), (D) glutathione (GSH), (E) superoxide dismutases (SOD) in colon tissue; (F) Effects of Sal B on reactive oxygen species (ROS) production in RAW264.7 cells. Data are expressed as the mean ± SD; **P < 0.01 compared with the sham (colon tissue) or normal (cell) group; ##P < 0.01 compared with the TNBS (IL-1β) control group (n = 7). Data in the normal or sham group is set to 100%; other data are the relative values compared with those in the sham group. SASP, sulfasalazine; Sal B (L), 20 mg/kg Sal B; Sal B (H), 80 mg/kg Sal B.
Figure 10
Figure 10
Analysis of colitis recurrence induced by IL-1β (10 ng/mL). (A) HE-staining analysis of colitis recurrence induced by IL-1β (10 ng/mL) after SASP or Sal B treatment in rat colonic tissue. Effects of Sal B on (B) macroscopically visible damage, (C) colon weight-to-length ratio, (D) myeloperoxidase (MPO), (E) proinflammatory cytokine TNF-α. Data are expressed as the mean ± SD; **P < 0.01 compared with as indicated (n = 10). Data in the sham group is set to 100%; other data are relative values. SASP, sulfasalazine; Sal B(L), 20 mg/kg Sal B; Sal B(H), 80 mg/kg Sal B.
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