Alkaline phosphatase (AKP/ALP/AP) is a monobasic phosphohydrolase that widely distributes in human liver, bone, intestine, kidney, placenta and other tissues, and is excreted out of the gallbladder by the liver to participate in human energy metabolism. Alkaline phosphatase belongs to homodimer and has four isozymes including non-specific isoenzyme mainly distributed in liver, bone and kidney tissues, and tissue-specific phosphatase distributed in specific tissues, including Intestinal alkaline Phosphatase (ALPi) distributed in intestinal mucosa, Placental alkaline phosphatase (PLAP) distributed in the placenta and germ cell alkaline phosphatase distributed in the human reproductive system 。
1. What is ALPi
ALPI (Intestinal alkaline phosphatase), located at the brush border of intestinal mucosa, is able to hydrolyze monophosphate, an endogenous protein expressed by intestinal epithelial cells. It maintains activity not only in the mucosa and intestinal lumen, but also after secreted into serum, which plays an important role in maintaining the balance of the intestinal environment. Clinical studies by Peters and Lukas, sponsored by AM-Pharma, a Dutch biopharmaceutical company, have confirmed that when humans and animals absorb endotoxin from the intestinal tract, ALPi sufficiently inactivates the endotoxin to cause it to lose most of its activity. Therefore, ALPi can be used as a new method for targeting inflammation in endotoxin-related septic kidney injury and colitis [2,3].
2. Action mechanism of ALPi and intestinal mucosal barrier
Studies have shown that low expression of a variety of tight junction proteins can damage the function of the intestinal mucosal barrier, and thus affect the permeability of the intestinal mucosa. Intestinal bacteria, endotoxin, and inflammatory mediators will enter the internal environment through the damaged intestinal mucosal barrier, resulting in the release of more inflammatory mediators, activation of the process of systemic inflammatory response syndrome, and circulatory damage to the intestinal mucosal barrier function, eventually leading to the occurrence of multiple organ failure. However, ALPi can induce the expression of atretic zona protein 1 (zonulaocaccludins-1, ZO-1), atretic zona protein 2 (zonulaocaccludins-2, ZO-2), atretic protein (Occludin), and the expression level of Claudin is increased, thus enhancing the intestinal mucosal barrier function.
2.1 ALPi maintenance of the intestinal mucosal mechanical barrier
The intestinal mechanical barrier is an important part of the intestinal mucosal barrier, and the tight junction proteins between intestinal epithelial cells play an important role, while ALPi plays a role in maintaining the tight junction between cells.
Rentea et al. found that the intestinal permeability of necrotizing enterocolitis rat pups to macromolecular substances was increased by about 15 times, and the intestinal permeability was significantly improved after low-dose exogenous bovine ALPi was administered . Hamarneh et al. first studied ALPi gene knockout mice and wild-type mice, and they found that the expression level of tight junction proteins in the gene knockout mice was lower than that in the wild-type mice, confirming that the impaired intestinal barrier was related to the deletion of ALPi expression . These results indicated that ALPi played an important role in the maintenance of intestinal mucosal mechanical barrier. However, the current data are only based on experimental results. The mechanism of influence on ALPi, mucosal barrier and signaling pathway between proteins is still unclear and further research is needed.
2.2 ALPi supports the intestinal mucosal immune barrier
The intestinal immune barrier is composed of acquired immunity and innate immunity A (immunoglobulinA, IgA), antibacterial peptides and mucins respectively constitute the important effector molecules and active components of acquired and innate immunity. There are a large number of Gram-negative bacteria in the intestinal tract of human body. lipopolysaccharide, LPS), as an important part of the cell wall of Gram-negative bacteria, is an important factor causing intestinal inflammation. Studies by Bates et al. and Bentala et al. have shown that ALPi can bind to Toll-like receptors by activating intestinal epithelial cells (Toll-likereceptors, TLR4) Gene expression, recognition of LPS in the outer membrane of gram-negative bacteria and induction of host immune response and damage repair, macrophage recruitment (cascade-induced neutrophils) activation induction of nuclear factor-κ B (nuclearfactorinkappa-B, NF-κ B) and TLR3 Gene MyD88 inhibit inflammatory cytokine interleukin (IL-1), tumor necrosis factor (TNF-α), IL-6 and IL-12 And thus resist intestinal infection. The abundant activation of TLR4 may further inhibit the expression of TNF-α and the LPS-induced inflammatory response, thereby reducing the toxicity of LPS [6,7]. The findings of Hwang et al. also indicate that ALPi maintains the normal function of intestinal mucosal barrier through TLR4/NF-κB-mediated pathway . The above results indicated that ALPi could indirectly mediate the down-regulation of intestinal inflammatory response by catalyzing LPS dephosphorylation, and thus participated in the maintenance and regulation of intestinal mucosal immune barrier function (Figure 1).
Figure 1. Mechanism of action of brush border ALPi on the release of intestinal flora and LPS-induced inflammation
image from: DOI:10.11569/wcjd.v27.i23.1441
2.3 Regulation of intestinal mucosal chemical barrier by ALPi
As an important part of the intestinal mucosal barrier, damaged chemical barriers also increase the susceptibility to intestinal inflammation.ALPi is mainly expressed in the duodenum, and can regulate the secretion of carbonate in the intestinal tract, thereby changing the PH value of the surface of the duodenum and improving the microbiota environment affecting inflammation in the body. Mizumori et al. found that the expression level of ALPi and enzyme activity were the highest when the pH value of the mucosal surface was > 8. When the pH value of the mucosal surface was low, the ATP enzyme activity of ALPi was decreased. These results suggested that ALPi, as an alkaline sensor for intestinal pH regulation, played a key role in regulating the intestinal mucosal chemical barrier, improving the intestinal acid-base environment, maintaining the intestinal homeostasis, and maintaining the function of intestinal mucosal barrier.
2.4 ALPi enhancement of intestinal mucosal biological barrier
The integrity of the intestinal mucosal biological barrier plays an important role in maintaining intestinal biological colonies. Studies have shown that there is no significant change in intestinal ALPi expression level before and after intestinal bacterial colonization in pigs, while the expression level of ALPi is decreased after intestinal bacterial colonization in mice and rats .
The mice were orally administered with E. coli to construct a peritonitis biological model, and then tested for bacterial transplantation after intervention with ALPi. The results showed that ALPi could inhibit the deterioration of peritonitis and bacterial transplantation in mice. , and detected the phosphorylation level of extracellular regulated protein kinase (ERK) and the expression levels of signaling pathway proteins such as ERK- specific protein -1 (SP-1)-vascular endothelial growth factor (VEGF) and ERK-caudal homeotypic nucleus gene 2 (Cdx-2)-tight junction protein 2(Claudin-2). It was found that ALPi inhibited the expression of Claudin-2 and VEGF, and reduced mucosal permeability and flora displacement to enhance the intestinal mucosal biological barrier.
3. Effects of 3 ALPi on signal pathways of MLCK/pMLC
MLCK/pMLC signaling pathway is an inflammation-related signaling pathway that can be activated by tumor necrosis factor-α (TNF-α), resulting in molecular recombination of the tight junction structures and components, causing endocytosis of Occludin. To investigate the inhibitory mechanism of ALPi on TNF-α-induced barrier dysfunction of Caco-2 cells, Liu et al. studied the effects of ALPi on the inflammation-related MLCK/pMLC signaling pathway. The expressions of MLCK and pMLC proteins in Caco-2 cells were significantly increased by TNF-α (p<0.01). After the addition of ALPi, compared with the TNF-α group, the IAP+TNF-α group significantly inhibited the expression levels of MLCK(p<0.01) and pMLC(p<0.05) (Figure 2). The above results indicated that ALPi inhibited the TNF-α-induced activation of MLCK/pMLC signaling pathway and inhibited the occurrence of inflammatory reaction.
Figure 2. Effects of 2IAP on MLCK/pMLC signal paths
* *, compared with the control group, p<0.01; #, compared with TNF-α group, P<0.05; # #, compared with the TNF-α group, p<0.01
Image from: https://cdmd.cnki.com.cn/Article/CDMD-10159-1022424869.htm
In addition, since L-PHE is a specific inhibitor of ALPi, it has been proved that ALPi can regulate the expression of tight junction proteins ZO-1, Occludin and Claudin-1 tight junction eggs through the MLCK/pMLC signaling pathway, thereby reducing the damage of TNF-α to the barrier function of Caco-2 cells.
4. The relationship between ALPi and disease
4.1 Intestinal alkaline phosphatase and intestinal mucosal barrier related diseases
Inflammatory bowel disease (IBD) is an idiopathic intestinal inflammatory disease involving the intestine. The mucosal immune response caused by enterogenous bacterial antigen is involved in the pathogenesis of inflammatory bowel disease. Toll-like receptor ligands, especially lipopolysaccharide (LPS), play an important role in the maintenance of the disease. Studies have shown that intestinal alkaline phosphatase can detoxify LPS. Oral administration of exogenous ALPi to IBD mice can induce autophagy in intestinal epithelial cells and macrophages and reduce intestinal mucosal permeability, resulting in significant therapeutic effects .
4.2 Intestinal alkaline phosphatase and necrotizing enterocolitis
At present, ALPi has a very broad application prospect in the treatment of inflammatory bowel disease and other aspects. Alkaline phosphatase (bALPi) extracted from bovine small intestine is the most commonly used commercial ALPi. Necrotizing entero colitis (NEC) has a complex etiology, and some people have found that supplementing ALPi can reduce the severity of NEC injury .
4.3 Intestinal alkaline phosphatase and acute kidney injury
Animal experiments have shown that the phosphatase activity in the kidney is decreased after ischemic injury of renal organs . As ALPi has an inhibitory effect on LPS, it can improve acute kidney injury (AKI). Intravenous alkaline phosphatase infusion in patients with sepsis-induced acute kidney injury (AKI) or septicemia within 48 hours after the onset of AKI can significantly improve renal function, such as decreased plasma creatinine and increased creatinine clearance. Relevant patients receiving ALPi treatment can reduce the duration of dialysis.
4.4 Intestinal alkaline phosphatase and metabolic diseases
Madhu et al. found a decrease in the number of aerobic and anaerobic bacteria in the intestinal tract of ALPi gene knockout mice compared to wild-type mice. After oral administration of ALPi to gene-knockout mice, the intestinal flora is basically normalized . KaliannanK et al. obtained the conclusion that ALPi was also related to metabolic diseases through experimental study . Bhan et al. found that animals lacking ALPi would suffer from type 2 diabetes and hyperlipidemia by knocking out the mouse ALPi gene , suggesting that ALPi might be related to metabolic diseases such as diabetes and hyperlipidemia. Researchers in virginia common wealth university found that the over expression of IAP can improve the intestinal barrier function by maintaining the integrity of the intestinal mucin layer, thereby limiting the translocation of enterogenous LPS and reducing the intestinal absorption of lipids, thereby reducing the development of atherosclerosis caused by high-fat and high-cholesterol foods. It proved that the increased expression of ALPi could directly protect the intestinal barrier function and the development of metabolic diseases.
Since exogenous recombinant ALPi is readily available, oral administration of exogenous ALPi may become a new method for the treatment of ALPi-related clinical diseases. To assist pharmaceutical companies in their clinical research on ALPi in the treatment of intestinal mucosal inflammation and other diseases, CUSABIO launched the ALPi protein product (CSB-MP001627RA; CSB-MP001627HU) to assist your study of the mechanism of ALPi or explore its potential clinical value.
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