Improving Small Intestinal Motility in Experimental Acute Necrotising Pancreatitis by Modulating the CPI-17/MLCP Pathway Using Chaiqin Chengqi Decoction

Abstract

Protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), a specific inhibitor of myosin light-chain phosphatase (MLCP) regulated by proinflammatory cytokines, is central for calcium sensitisation. We investigated the effects of chaiqin chengqi decoction (CQCQD) on the CPI-17/MLCP pathway in the small intestinal smooth muscle cells (SMCs) and strips (SMS) in an AP model. Necrotising AP was induced in rats by intraperitoneal injections (IPI) of L-ornithine (3.0 g/kg, pH 7.0; hourly × 2) at 1 hour apart; controls received saline. In treatment groups, carbachol (CCh; 60 μg/kg, IPI) or CQCQD (20 g/kg; 2-hourly × 3, intragastric) was administered. The necrotising AP model was associated with systemic inflammation (serum IL-1β and TNF-α) and worsened jejunum histopathology and motility (serum vasoactive intestinal peptide and intestinal fatty acid-binding protein) as the disease progressed. There was decreased intracellular calcium concentration ([Ca²⁺]_i) SMCs. Contractile function of isolated SMCs was reduced and associated with down-regulated expression of key mRNAs and proteins of the CPI-17/MLCP pathway as well as increased IL-1β and TNF-α. CQCQD and CCh significantly reversed these changes and the disease severity. These data suggest that CQCQD can improve intestinal motility by modulating the CPI-17/MLCP pathway in small intestinal smooth muscle during AP.

Figure 1

Time-course intestinal histopathology changes in L-ornithine-induced acute necrotising pancreatitis. Rats received 2 intraperitoneal injections of L-ornithine (Orn; 3.0 g/kg; pH 7.0) at 1 hour apart and controls received the same regimen of normal saline injections. Rats were sacrificed at 12, 24, 36, and 48 hours to assess severity of acute pancreatitis. (a) Representative H&E sections of upper jejunum (100x). (b) Intestinal histopathology scores: A, overall; B, crypt damage; C, inflammation severity; D, inflammation extent. ^∗P < 0.05vs. saline group; ^†P < 0.05vs. L-ornithine 12 h group. Values are means ± SEM of 4–6 animals per group.

Figure 2

Effects of CQCQD and CCh on intestinal histopathology and serum intestinal motility biomarkers. Rats received 2 intraperitoneal injections of L-ornithine (Orn; 3.0 g/kg; pH 7.0) at 1 hour apart and controls received the same regimen of normal saline injections. In the treatment groups, rats either received single intraperitoneal injection of carbachol (CCh; 60 μg/kg) or 3 times oral gavage of chaiqin chengqi decoction (CQCQD; 20 g/kg) at 2-hourly interval begun at 24 hours after the first L-ornithine injection. Rats were sacrificed at 30 hours to assess severity of acute pancreatitis. (a) Representative H&E sections of upper jejunum (100x). (b) Intestinal pathology scores: A, overall; B, crypt damage; C, inflammation severity; D, inflammation extent. (c) Serum vasoactive intestinal peptide (VIP). (d) Serum intestinal fatty acid-binding protein (iFABP). ^∗P < 0.05vs. saline group; ^†P < 0.05vs. L-ornithine group. Values are means ± SEM of 5–10 animals per group.

Figure 3

Effects of CQCQD and CCh on [Ca²⁺]_i in cells and contraction in strips of jejunum smooth muscles. Rats received 2 intraperitoneal injections of L-ornithine (Orn; 3.0 g/kg; pH 7.0) at 1 hour apart and controls received the same regimen of normal saline injections. In the treatment groups, rats either received single intraperitoneal injection of carbachol (CCh; 60 μg/kg) or 3 times oral gavage of chaiqin chengqi decoction (CQCQD; 20 g/kg) at 2-hourly interval begun at 24 hours after the first L-ornithine injection. Rats were sacrificed at 24 or 30 hours to isolate jejunum smooth muscle cell and strips for assessing intracellular calcium concentrations [Ca²⁺]_i and contraction, respectively. (a) Representative confocal microscopy images of Fluo 4-AM stained (×200). The lower left corner for each image is a representative morphological observation field of isolated smooth muscle cell (200x). (b) Fluo 4-AM fluorescence intensity (FI) for all experimental groups. (c) Example images of jejunum smooth muscle strip contraction for each experimental group. X axis represents time in seconds and Y axis represents (g) (d) Contraction tension. (e) Contraction frequency. All data are normalised according to saline injection group using arbitrary unit (AU). The bar of medium and sparse patterns represents rats sacrificed at 24 and 30 hours after the first L-ornithine/saline injection, respectively. ^∗P < 0.05vs. saline group; ^†P < 0.05vs. L-ornithine group. Values are means ± SEM of 4 animals per group.

Figure 4

Effects of CQCQD and CCh on mRNA and protein expression of IL-1β, TNF-α, and CPI-17 in jejunum smooth muscle strips. Rats received 2 intraperitoneal injections of L-ornithine (Orn; 3.0 g/kg; pH 7.0) at 1 hour apart and controls received the same regimen of normal saline injections. In the treatment groups, rats either received single intraperitoneal injection of carbachol (CCh; 60 μg/kg) or 3 times oral gavage of chaiqin chengqi decoction (CQCQD; 20 g/kg) at 2-hourly interval begun at 24 hours after the first L-ornithine injection. Rats were sacrificed at 24 or 30 hours to isolate jejunum smooth muscle strips. (a) Representative images of agarose gel electrophoresis of targeted RT-PCR mRNAs. (b) Histograms for semiquantitative mRNA expression. (c) Representative western-blotting images of proteins. (d) Histograms for semiquantitative protein expression. (e) Example images of immunofluorescence (s, smooth muscle layer; m, mucosa; sm, submucosa). Relative expression of individual mRNA and protein are calculated according to β-actin. The bar of medium and sparse patterns represents rats sacrificed at 24 and 30 hours after the first L-ornithine/saline injection, respectively. ^∗P < 0.05vs. saline group, ^†P < 0.05vs. L-ornithine group; ^‡P < 0.05vs. CCh-treated group. Values are means ± SEM of 4 animals per group.

Figure 5

Effects of CQCQD and CCh on mRNA and protein expression of p-CPI-17, p-MYPT1, and p-MLC20 in jejunum smooth muscle strips. Rats received 2 intraperitoneal injections of L-ornithine (Orn; 3.0 g/kg; pH 7.0) at 1 hour apart and controls received the same regimen of normal saline injections. In the treatment groups, rats either received single intraperitoneal injection of carbachol (CCh; 60 μg/kg) or 3 times oral gavage of chaiqin chengqi decoction (CQCQD; 20 g/kg) at 2-hourly interval begun at 24 hours after the first L-ornithine injection. Rats were sacrificed at 24 or 30 hours to isolate jejunum smooth muscle strips. (a) Representative images of agarose gel electrophoresis of targeted RT-PCR mRNAs. (b) Histograms for semiquantitative mRNA expression. (c) Representative western-blotting images of proteins. (d) Histograms for semiquantitative protein expression. (e) Example images of immunofluorescence (s, smooth muscle layer; m, mucosa; sm, submucosa). ^∗P < 0.05vs. saline group, ^†P < 0.05vs. L-ornithine group, and ^‡P < 0.05vs. CCh-treated group. Values are means ± SEM of 4 animals per group.

Figure 6

Summary diagram of the role of CQCQD in early stage of acute pancreatitis. SIRS, systemic inflammatory response syndrome; MODS, multiple organ dysfunction syndrome.