Lactoferrin Reduces Necrotizing Enterocolitis Severity by Upregulating Intestinal Epithelial Proliferation

Abstract

Introduction Necrotizing enterocolitis (NEC) is a devastating intestinal illness in premature infants characterized by severe intestinal inflammation. Despite medical interventions, NEC mortality remains alarmingly high, which necessitates improved therapies. Lactoferrin is among the most abundant proteins in human milk and has important immunomodulatory functions. While previous studies have indicated protective effects of lactoferrin against neonatal sepsis and NEC, the underlying mechanism remains unclear. We hypothesize that lactoferrin downregulates inflammation and upregulates proliferation in intestinal epithelium during NEC injury. Materials and Methods NEC was induced by hypoxia, gavage feeding of hyperosmolar formula and lipopolysaccharide between postnatal day P5 and P9 (n = 8). Breastfed mice were used as control (n = 7). Lactoferrin (0.3 g/kg/day) was administered once daily by gavage from P6 to P8 in both NEC (NEC + Lac; n = 9) and control mice (Cont + Lac; n = 5). Distal ileum was harvested on P9 and analyzed for disease severity, inflammation, and proliferation. Groups were compared using one-way ANOVA and t-test appropriately; p < 0.05 was considered significant. Results Compared to NEC group, lactoferrin-treated NEC mice had reduced disease severity, reduced inflammation markers IL-6 and TNF-α expression and increased intestinal stem cell marker Lgr5 + expression. Lactoferrin-treated NEC mice exhibited increased nuclear β-catenin, indicating upregulated Wnt pathway, and increased Ki67 positivity, suggesting enhanced proliferation. Furthermore, lactoferrin administration to control mice did not affect intestinal inflammation as well as Lgr5 + stem cell expression and epithelial proliferation. This supports the safety of lactoferrin administration and indicates that the beneficial effects of lactoferrin are present when intestinal injury such as NEC is present. Conclusion Lactoferrin administration reduces the intestinal injury in experimental NEC by downregulating inflammation and upregulating cell proliferation. This beneficial effect of lactoferrin in stimulating cell proliferation is mediated by the Wnt pathway. This experimental study provides insights on the mechanism of action of lactoferrin in NEC and the role of lactoferrin in enteral feeding.

Enteral Feeding Interventions in the Prevention of Necrotizing Enterocolitis: A Systematic Review of Experimental and Clinical Studies

“…Third, it is challenging to determine the optimal therapeutic regimen (dose, frequency, timing). Even though dose is of clear importance for the therapeutic effect [36,101], most animal studies only test a single dose and frequency of administration and it is therefore unclear how the dose and administration regimen used in animal studies should be translated to the human neonate. Of note, the optimal dose for the human neonate may be very well dependent on individual baseline levels, e.g., an infant with baseline deficit of a specific nutritional component may benefit from a higher dosage than an infant with baseline values within the normal range.…”

“…These detrimental effects are likely caused by the high dose of the lactoferrin used, as in in vitro experiments with cultured intestinal epithelial cells a high dose, but not lower doses, of bovine lactoferrin upregulated the expression of pro-apoptotic proteins and HIF1α signaling pathway proteins and downregulated that of anti-apoptotic proteins and proteins related to cell proliferation [36]. In another study using a mouse model of NEC, enteral recombinant lactoferrin administration (6 g/L) prevented a NEC protocol induced decrease in Ki67 immunoreactivity, preserved beta-catenin immunoreactivity and restored LGR5 mRNA levels in the distal ileum [101]. Together, these studies demonstrate that the dose of the nutritional intervention studied is important and should be taken into account when designing a clinical trial.…”

“…Fish oil (rich in n-3 PUFA) [82,83] MPL [84] MFGM [48] Very low fat diet [85] Reduced long chain triacylglycerol diet (considered pre-digested) [85] Pomegranate seed oil [47] Carbohydrate/sugar-based interventions HMO [38,42,49,[86][87][88][89][90] Neutral HMO (no sialic acids) [42] −2 HMO (two sialic acids) [42] DSLNT (HMO) [42] DSLNnT (synthetic disialyl glycan) [89,90] DS'LNnT (synthetic disialyl glycan) [89,90] 2 -FL [87,91,92] 6 -SL [92] 2 -FL and 6 -SL [42,92] Sialylated HMO [93] Sialylated GOS [87] GD3 [50] Hyaluronan 35 kD [94] Protein/amino acid-based interventions L-Glutamine/glutamine [84,[95][96][97] Arginine [98,99] L-Carnitine [99] N-Acetylcysteine [85] Lactadherin [51] OPN [100] Lactoferrin [101] IAP [102,103] [104] HB-EGF [41,[58]…”

“…In preterm pigs, enteral bovine lactoferrin administration reduced proximal intestinal IL1β, but not IL8, protein levels [35]. Terminal ileum mRNA expression levels of IL6 and TNFα were reduced by enteral feeding supplemented with lactoferrin in a murine NEC model [101]. Oral administration of N-acetylcysteine reduced intestinal mRNA levels of IL1β and TNFα [85].…”

Inhibition of A2B Adenosine Receptor Attenuates Intestinal Injury in a Rat Model of Necrotizing Enterocolitis

“…All these changes induced epithelial cell edema, apoptosis, and even death. It has been reported that the enhanced epithelial cell proliferation was associated with an alleviated NEC severity [ 38 ]. The proliferated epithelial cells contributed mostly in the repair of the impaired mucosa barrier, and we observed increased epithelial cell proliferation in NEC rats after PSB1115 administration.…”