Intestinal Mucosal Barrier Bodyguard --ALPi

1. What is ALPi?

ALPi, also known as IAP, is a brush border enzyme primarily produced by enterocytes (that line the small intestine) and then secreted into the lumen, blood, and stool.

ALPi hydrolyzes phosphate from the lipid A moiety of lipopolysaccharides (LPS) and thus significantly reduces LPS pro-inflammatory activity ^[4]. In addition to detoxifying LPS, ALPi also protects gut barrier integrity, regulates gut microbiota, dephosphorylates pro-inflammatory nucleotides, and modulates bicarbonate secretion and duodenal surface pH ^[4-6]. ALPi is essential for intestinal homeostasis and microbial balance.

2. Mechanisms of ALPi in Protecting the Intestinal Mucosal Barrier

ALPi plays a critical role in maintaining the integrity and functionality of the intestinal mucosal barrier. Its mechanisms of action are multifaceted, mainly involving regulation of tight junction proteins, LPS detoxification, attenuation of inflammation, and maintenance of gut microbial homeostasis.

2.1 Regulation of Tight Junction Proteins by ALPi

The intestinal mucosal barrier relies on tight junction proteins (TJPs) such as ZO-1, ZO-2, Occludin, and Claudins to maintain epithelial integrity. These TJPs regulate paracellular permeability and prevent the translocation of harmful substances. ALPi upregulates the expression of these tight junction proteins (TJPs), thus maintaining tight junction integrity and reducing gut permeability (leaky gut) ^[8]. Additionally, IAP preserves the localization of these proteins during inflammation, further strengthening barrier function ^[5].

Animal studies demonstrated that ALPi deficiency impairs the barrier. In necrotizing enterocolitis (NEC) rat models, exogenous bovine ALPi reduced permeability by up to 15-fold ^[7]. Knockout mice lacking ALPi had reduced expression of TJPs, including ZO-1, ZO-2, and occludin, and claudin 1 when compared to wild-type mice ^[9]. The administration of exogenous ALPi supplementation was demonstrated to prevent alcohol-induced intestinal barrier dysfunction and TJP loss ^[10].

ALPi also alleviates inflammation-caused damage to TJPs by detoxifying lipopolysaccharides (LPS) and reducing pro-inflammatory cytokine levels. This protective effect helps preserve tight junction structure and function ^[11,12].

2.2 Detoxification of LPS by ALPi

ALPi is a key enzyme that detoxifies LPS, a pro-inflammatory component of Gram-negative bacteria. ALPi detoxifies LPS, thereby inhibiting downstream activation of immunocytes (TNF-α, IL-1β, IL-6, IL-12) and the subsequent inflammatory responses through TLR4 signaling ^[13]. Hwang et al. also indicated that ALPi maintains the normal function of the intestinal mucosal barrier through the TLR4/NF-κB-mediated pathway ^[14].

Figure 1. Detoxification of LPS by ALPi

The picture is cited from：https://pmc.ncbi.nlm.nih.gov/articles/PMC9026380/

LPS detoxification is critical to prevent endotoxemia, a condition where LPS translocates from the gut into the bloodstream, causing systemic inflammation and organ dysfunction ^[15].

2.3 Anti-Inflammatory Effect of ALPi

During inflammation, extracellular purines (e.g., adenosine, ADP, ATP) and pyrimidines (e.g., UDP, UTP) released from host cells and gut microbiota activate purinergic receptors autocrinally/paracrinally ^{[16]. Extracellular ATP is hydrolyzed by ALPi to ADP, AMP, and adenosine [17], which then binds adenosine receptors (A1, A2A, A2B, A3) to provide intestinal protection and anti-inflammatory effects [18]. M. Estaki et al. showed that oral ALPi alleviates metabolic syndrome in ALPi-knockout mice fed a high-fat diet [19].}

ALPi exerts anti-inflammatory effects by dephosphorylating pro-inflammatory molecules like ATP and flagellin ^[17], thereby reducing activation of inflammatory pathways such as the NF-κB signaling pathway, which are involved in gut barrier dysfunction ^[6]. Orally administered ALPi has been shown to decrease levels of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-8, thus alleviating intestinal inflammation ^[12][17].

2.4 Modulation of Intestinal Microbiota

ALPi activity is closely tied to microbiota composition. In murine models, ALPi deficiency leads to dysbiosis characterized by an overgrowth of harmful bacteria ^[21]. Conversely, oral ALPi supplementation restores microbial balance and ameliorates colitis symptoms ^[8]. By shaping microbial ecology, ALPi helps maintain symbiosis and prevents the cascade of chronic intestinal inflammation.

ALPi helps maintain gut microbial homeostasis by promoting mucosal tolerance to gut bacteria through LPS detoxification ^[15]. ALPi-knockout mice show increased bacterial translocation after intestinal injury ^[20] and reduced bacterial diversity, reversible with ALPi supplementation ^[21]. ALPi supports bacterial growth by dephosphorylating luminal ATP (which inhibits Gram-positive bacteria) ^[22,23]. Exogenous ALPi administration alleviates Salmonella and C. difficile infections by restoring normal gut microbiota ^[24].

Additionally, ALPi functions as an alkaline sensor in the duodenum, regulating bicarbonate secretion and mucosal pH. Optimal ALPi activity occurs at mucosal pH >8. Acidic environments reduce its enzymatic function, thereby impairing the intestinal mucosal barrier ^[25]. Thus, ALPi contributes to maintaining a balanced pH, supporting microbial homeostasis and mucosal protection.

3. ALPi and Disease Associations

ALPi is a critical enzyme that plays a significant role in maintaining intestinal homeostasis and barrier function. Its deficiency or altered activity has been implicated in various intestinal diseases, ranging from inflammatory conditions to dysbiosis and increased permeability.

3.1 Inflammatory Bowel Disease (IBD)

IBD pathogenesis involves aberrant immune responses to gut bacteria. Studies have shown that ALPi deficiency or reduced activity is linked to increased intestinal inflammation, dysbiosis, and systemic inflammation, all of which are hallmarks of IBD ^[26]. ALPi detoxifies LPS, enhances autophagy in epithelial cells and macrophages, and reduces permeability. Oral administration of ALPi significantly alleviated colitis severity in mouse IBD models ^[27].

3.2 Necrotizing Enterocolitis (NEC)

NEC is a severe gastrointestinal condition primarily affecting preterm infants, often leading to high morbidity and mortality rates. LPS is a key mediator of inflammation in NEC. Studies have shown that preterm infants with NEC exhibit significantly lower ALPi levels compared to healthy controls, suggesting that ALPi deficiency may contribute to the development of NEC ^[28].

Additionally, ALPi has been found to promote autophagy, improve intestinal perfusion, and prevent dysbiosis, further protecting against NEC-related intestinal injury. These findings underscore the enzyme's potential as a preventive and therapeutic agent in NEC management ^[29,30].

3.3 Acute Kidney Injury (AKI)

Sepsis-induced AKI involves systemic inflammation and LPS toxicity. Clinical trials demonstrated that intravenous recombinant ALPi improved renal function, reduced plasma creatinine, and shortened dialysis duration in AKI patients ^[31].

3.4 Metabolic Disorders

ALPi-deficient mice exhibit dysbiosis, type 2 diabetes, and hyperlipidemia. Oral ALPi restores microbial balance and protects against metabolic syndrome ^[32]. Overexpression of ALPi in animal models preserved the mucin layer, reduced LPS absorption, and decreased atherosclerosis progression ^[33]. These findings suggest ALPi plays a crucial role in metabolic health.

4. Clinical and Translational Perspectives

Recent Gut and JCI publications highlight recombinant ALPi as a therapeutic candidate for multiple diseases, including IBD, AKI, and metabolic syndrome. AM-Pharma has advanced recombinant human ALPi (recAP) into clinical trials, showing safety and efficacy in sepsis-related AKI ^[31]. Furthermore, the role of ALPi in microbiome regulation is an emerging area, connecting gut health to systemic diseases such as neuroinflammation and cardiovascular disorders.

5. Research Reagents for ALPi

Specific antibodies, recombinant ALPi proteins, and ELISA kits are essential to support ongoing research. These reagents enable:

• Tight junction protein analysis (ZO-1, ZO-2, Claudin, Occludin).
• LPS detoxification assays for immune modulation studies.
• Barrier permeability experiments in Caco-2 and organoid models.
• Biomarker detection in IBD, NEC, AKI, and metabolic disorder studies.

CUSABIO has developed a comprehensive portfolio of ALPi-related products, including recombinant ALPi proteins, ALPi polyclonal antibodies, and sensitive ALPi ELISA kits. These reagents provide reliable tools for exploring ALPi function, signaling pathways, and therapeutic potential.

Conclusion

ALPi stands as a critical enzymatic defender of the gut mucosal barrier, safeguarding intestinal integrity by detoxifying endotoxins, regulating immune responses, and preserving tight junction function. Its relevance in diverse inflammatory and metabolic pathologies underscores its promising potential as a biomarker and therapeutic agent. Cutting-edge ALPi antibodies, recombinant proteins, and ELISA kits now empower researchers to dissect its role with precision and accelerate discoveries toward novel gut-targeted interventions.