Molecular Regulation Mechanism Of Chang’an Ⅱ Decoction On Tight Junctions Of Intestinal Epithelial Barrier Via MLCK-MLC Signaling Pathway

BackgroundIrritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by abdominal discomfort and altered bowel habits in the absence of structural or biochemical evidence accounting for these symptoms, and diarrhea-predominant IBS (IBS-D) is the major type encountered in clinics.The etiopathogenesis and pathophysiological mechanism of IBS are not completely illuminated. Recently, more and more evidence has shown that altered epithelial tight junctions and increased permeability participate in the occurrence and development of IBS-D, in which proinflammatory cytokines released by the immune system may increaseIntestinal permeability directly by down-regulating the relevant proteins of tight junctions. Tumor necrosis factor (TNF-a) plays a key role in the complex chain reaction mediating intestinal barrier dysfunction. It is well known that TNF-a disrupts Intestinal barrier function largely by up-regulating myosin chain kinase (MLCK), which leads to increased myosin light chain (MLC) phosphorylation. This phosphorylation then leads to activation of ATPase, which provides the metabolic energy required for the mechanical contraction of actin and myosin filaments and further remodels the TJs. Furthermore, TNF-a can up-regulate other proinflammatory cytokines to form cascade effects on the injury of intestinal epithelial cells.So far, there are no targeted regulatory drugs that repair TJs injury triggered by proinflammatory cytokines. Our preliminary research has shown that Chang’anⅡ decoction can down-regulate the expression of TNF-a and IL-1β, lower paracellular permeability and repair intestinal barrier dysfunction in a IBS-D animal model with disease-syndrome integration. However, the probable molecular mechanism underlying these effects is still unknown.ObjectiveTo investigate the influence of Chang’an II decoction on the intestinal epithelial TJs injury induced by TNF-a, and to further explore the probable underlying molecular mechanism of this effect via MLCK-MLC signaling pathway and imbalanced shift of cytokines.Methods1. Caco-2 cells were plated on collagen-coated permeable polycarbonate membrane Transwell supports and grown as monolayers for 21 days. Subsequently, the integrity of monolayers were estimated with transepithelial electrical resistance (TER), alkaline phosphatase(AP) activity, paracellular permeability, apparent permeability coefficients(Papp) of FD-4 and ultrastructure of tight junctions. Next, different concentrations of TNF-a (0 ng/mL,1 ng/mL,10 ng/mL,100 ng/mL) were used to induce intestinal epithelial TJs injury for 24 h,48 h and 72 h. The dynamic measurements of TER were to filtrate the optimum dose and action time.2. For the experiments, cells were divided into six groups as follows:normal group(treated with medium plus 10% control serum), model group(treated with 30 ng/mL TNF-a and medium plus 10% control serum), Chang’an Ⅱ-H group(treated with 30 ng/mL TNF-a and medium plus 10% high dose of Chang’an II decoction serum), Chang’an Ⅱ-M group(treated with 30 ng/mL TNF-a and medium plus 10% middle dose of Chang’an II decoction serum), Chang’an Ⅱ-L group(treated with 30 ng/mL TNF-a and medium plus 10% low dose of Chang’an Ⅱ decoction serum). As the positive drug group, berberine group was treated with 30 ng/mL TNF-a and medium plus 10% control serum with berberine at 100 μM. The observation of TER, paracellular permeability and ultrastructure of tight junctions by TEM were used to investigate the influence of Chang’an II decoction on the intestinal epithelial TJs injury.3. To investigate the probable underlying molecular mechanism via MLCK-MLC signaling pathway, the distribution of tight junction proteins ZO-1, Claudin-1 and F-actin was analyzed by immunofluorescence. The translocation of NF-kB p65 to nuclei was analyzed by immunoflurescence. The expression of MLCK mRNA was detected by Real-Time polymerase chain reaction(PCR).The expressions of ZO-1, Claudin-1, Occludin, MLCK, MLC and p-MLC were determined by western-bloting.4. To study the influence of Chang’an Ⅱ decoction on the imbalanced shift of cytokines induced by TNF-a, cytokines in the culture supernatant were evaluated by ELISA.Results1. Establishment and evaluation of intestinal epithelial TJs injuryThe results showed that TER of monolayers has been increasing gradually. After 21 days of in vitro culture, the TER reached a relatively stable value of (1568.08±206.46) Ω·cm2, the paracellular permeability of FD-4 was (2.31 ±1.12)%, the Papp was (1.95±0.9)X 10-7cm · s-1 and the AP activity in the apical side was significantly higher than that in the basolateral side. Also, TEM showed that the microvillis and continuous TJs were differentiated in the apical side of intestinal epithelial cells.The dynamic TER showed that TER of normal group has been increasing gradually until 72 h. To the contrast, TERs of TNF-a 1 ng/mL group,10 ng/mL and 100 ng/mL group decreased gradually until 24 h, and then increased until 72 h. At the end of 24 h, compared with normal group, TERs of TNF-a 1 ng/mL group,10 ng/mL and 100 ng/mL group were significantly decreased (p<0.05), especially for the 10 ng/mL and 100 ng/mL group (p<0.05).However, there was no statistical difference between the two groups (p>0.05).2. The influence of Chang’an Ⅱ decoction on the intestinal epithelial TJs injury induced by TNF-a(1) TER was maintained in the normal group during the experiment. In contrast, the model group experienced a progressive decrease in TER. However, the TER decrease induced by TNF-a was significantly inhibited by Chang’an II decoction and barberine in a dose-dependent manner. The TERs of all the drug treated groups increased initially and peaked at 6 h, and then subsequently decreased, but they were higher than that of the model group. It is worth mentioning that the Chang’an Ⅱ-H group played the role in the earliest and longest.Compared with the normal group. FD-4 flux was significantly increased after treated with TNF-α for 24 hours(p<0.05). In contrast, the increased paracellular permeability induced by TNF-a was significantly inhibited by Chang’ an Ⅱ decoction and berberine(p<0.05). There is no obvious statistical difference among all the drug treated groups(p>0.05).(2) The ultrastructure showed that in the control group, the TJs of Caco-2 monolayers were intact and continuous. Meanwhile, the microvillis on the surface of the cell membrane were well-distributed and regularly-arrayed. However, the TJs of the model group were incomplete, fractional and loose. In addition, the gap junctions were obviously widened and the number of desmosomes w decreased. At the same time, the microvillis on the surface of the cell membrane were uneven distributed and scattered. The ultrastructural injury of TJs induced by TNF-a restored in all drug-treated groups.3. The probable underlying molecular mechanism via MLCK-MLC signaling pathway(1) Chang’an II decoction repairs TNF-a-induced altered localization of ZO-1, Claudin-1 and the actin-cytoskeletonIn the control group, the immunofluorescences of ZO-1,Claudin-1 and F-actin were each localized to intercellular tight junctions, along the edges of the cells. Compared with the normal group, the fluorescence intensity of the model group was significantly weakened and irregular, and the fluorescence of Claudin-1 diffused to the cytoplasm. The fractured fluorescence signals of all drug treated groups were repaired, especially the Chang’an II-H group.Western-bloting results showed that there were no significant differences of Claudin-1 and Occludin between normal group and model group(p>0.05). The expression of ZO-1 in the model group was significant lower than that in the normal group, and only the Chang’an Ⅱ-H group can up-regulate the expression of ZO-1(p<0.05).(2) Chang’an Ⅱ decoction regulated the microfilament of cytoskeleton① NF-kB p65:In the control group, NF-kB p65 subunits were present mainly in the cytoplasm. In contrast, the model group experienced a mass translocation of NF-kB p65 into the nucleus. This nuclear translocation was accompanied by a corresponding decrease of NF-kB p65 in the cytoplasm. Compared with the model group, all drug-treated groups showed largely attenuated NF-kB p65 translocation into the nucleus.② MLCK mRNA:The expression of MLCK mRNA in the model group was significantly higher than that in the normal group(p<0.05). Compared with the model group, the expression of MLCK mRNA in all drug-treated groups was significantly down-regulated (p<0.05), and the berberine group had the lowest MLCK mRNA among all drug-treated groups (p<0.05).③ MLCK,MLC and p-MLC:The expression of MLCK,MLC and p-MLC in the model group was significantly up-regulated compared with the normal group (p<0.05). However, the expression of MLCK in all drug-treated groups was lower than that in the model group (p<0.05), especially in the Chang’an Ⅱ-H group (p<0.05). Compared with the model group, the expression of MLC in the Chang’an Ⅱ-H and berberine group was significantly reduced (p<0.05). Additionly, p-MLC expression in the Chang’an Ⅱ-H group was more down-regulated compared to that in the model group (p<0.05).4. ELISA results showed that IFN-y, IL-6 and IL-8 in the model group was significantly higher than that in the normal group (p<0.05). Compared with the model group, IFN-y, IL-6 and IL-8 in all drug-treated groups was significantly decreased (p<0.05). Meanwhile, Chang’an II-H group had the lowest IFN-y and IL-8 (p<0.05). IL-4 and IL-10 in model group were significantly lower than normal group (p<0.05). And compared with model group, IL-4 of Chang’an Ⅱ-L group and berberine group were obviously increased (p<0.05). Meanwhile, IL-10 of berberine group was only increased among all drug treated drugs (p<0.05).Conclusions1. Caco-2 cells plated on collagen-coated permeable polycarbonate membrane Transwell supports for 21 days can establish intestinal epithelial TJs, and TNF-a has a time-dependent and dose-dependent injury on TJs. Finally, the optimum dose is 30ng/mL and the action time is 24 h.2. Chang’an Ⅱ Decoction can improve the microvilli morphology and repair the TJs injury to decrease the paracellular permeability.3. Chang’an Ⅱ-H group can inhibite the up-regulation of MLC phosphorylation mediated by NF-kB p65 and MLCK, which might be the molecular mechanism involved in the protective action of Chang’an Ⅱ Decoction againstIntestinal epithelial barrier dysfunction triggered by TNF-a.4. Chang’an Ⅱ Decoction can down-regulate the expression of IFN-γ、IL-6 and IL-8 to rectify imbalanced shift of cytokine expression triggered by TNF-a, especially the Chang’an Ⅱ-H group.