A total of 60 newborn Sprague-Dawley (SD) rats (6–8 g; 30 Male, 30 Female) were obtained from pregnant SD rats (Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China) on day 21 of gestation. Thereafter, newborn rats were fed with 0.1 ml artificial milk (Pet-Ag, Inc., Hampshire, IL, USA) twice a day. Rats were housed at 30°C and 60% humidity with a 12-h light-dark cycle. NEC models were established via N₂ inhalation for 90 sec every 4 h and oral administration of 4 mg/kg/day lipopolysaccharides (LPS; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) on days 0 and 1 (20). The present study was approved by the Animal Ethics Committee of The Second People's Hospital of Liaocheng (Linqing, China).

Newborn were weighed and randomly divided into three groups (n=20). Rats in the normal control (control) group stayed with their mothers in the same cage following birth and were fed breast milk without any intervention. Neonatal rats in the NEC model (NEC) group were placed in an incubator 48 h after birth and established as the neonatal NEC rat models. Neonatal rats in the berberine intervention (NEC + berberine) group were established as NEC rat models and berberine (Sigma-Aldrich; Merck KGaA) was administered twice a day for 4 days by gavage at a dose of 0.6 g/kg/day dissolved in artificial milk.

Rats that developed distress (lethargy, abdominal distention and bloody diarrhea) or imminent death prior to 96 h were sacrificed to acquire the whole intestine. At 96 h later, remaining rats were sacrificed to obtain the intestines. Intestinal samples were fixed in 75% ethanol at 4°C for 16 h embedded in paraffin and sliced into 4–6-µm-thick sections. These sections were stained using hematoxylin and eosin both for 1 min at room temperature. Histological evaluation of NEC was performed using light microscopy (magnification, ×200) (Olympus BX51; Olympus Corporation, Tokyo, Japan). NEC severity was assessed according to the NEC scoring system (3). Histological changes in the intestinal architecture of rats with NEC were assigned an NEC grade: Grade N (normal), noseparation in the submucosa or lamina propria; grade L (low), slight submucosal and lamina propria separation; grade M (moderate), increased submucosal and lamina propria separation with edema of the submucosa; grade I (intermediate), severe separation of the submucosa and lamina propria; and grade S (severe), necrosis and loss of villi structure. Rats with grade M, I or S were deemed to have NEC.

The rat intestinal tissues were homogenized in NP40 lysis buffer (Sigma-Aldrich; Merck KGaA) on ice and the homogenates were quantified using a BCA assay (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Total intestinal proteins were assessed using an ELISA kit to evaluate the levels of IL-6 (cat. no. ab100712; Abcam, Cambridge, UK), IL-10 (cat. no. ab214566; Abcam), MUC2 (cat. no. LS-F4717; LifeSpan Biosciences, Inc., Seattle, WA, USA) and SIgA (cat. no. SE120114; Sigma-Aldrich; Merck KGaA) in the samples.

Total RNA was extracted from intestinal tissues using TRIzol Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA (1 µg) was used to generate cDNA with SuperScript II reverse transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR was performed in triplicate using SYBR Premix Ex Taq (Takara, Dalian, Liaoning, China) and SsoFast™ Probes Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and a standard thermocycling procedure (35 cycles) was performed on a Bio-Rad CFX96™ Real-time PCR System (Bio-Rad Laboratories, Inc.). The thermocycling conditions were as follows: 95°C for 2 min; followed by 35 cycles of 95°C for 1 min, 60°C for 1 min and 72°C for 1 min; then extension was performed at 72°C for 10 min. The 2^−ΔΔCq method was used to analyze the relative changes in gene expression (4). The primers used were as follows: TLR4 forward, 5′-GCATCATCTTCATTGTCCTTGA-3′ and reverse, 5′-CTTGTTCTTCCTCTGCTGTTTG-3′; NF-κB forward, 5′-ATGGCAGACGATGATCCCTAC-3′ and reverse, 5′-CGGAATCGAAATCCCCTCTGTT-3′; iNOS forward, 5′-GAGGCCCAGGAGGAGAGAGATCCG-3′ and reverse, 5′-TCCATGCAGACAACCTTGGTGTTG-3′; TNF-α forward, 5′-CCAGACCCTCACACTCAGATC-3′ and reverse, 5′-CACTTGGTGGTTTGCTACGAC-3′; and β-actin forward, 5′-CTAAGGCCAACCGTGAAAAG-3′ reverse, 5′-TACATGGCTGGGGTGTTGA-3′.

Total protein was extracted from ileal tissues with lysis buffer (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). The protein concentration was determined using a BCA assay. Proteins (50 µg/lane) were separated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane and blocked with 50 g/l skimmed milk at room temperature for 1 h. Primary antibodies against TLR4 (1:1,000; cat. no. ab13556), iNOS (1:1,000; cat. no. ab3523) and NF-κB (1:1,000; cat. no. ab32536; all Abcam) were incubated with the membranes overnight at 4°C, following which membranes were incubated with an horseradish peroxidase-conjugated antibody (1:5,000; cat. no. 7071; Cell Signaling Technology, USA) at room temperature for 1 h. The protein bands were visualized with a G-BOX imaging system (Syngene Europe, Cambridge, UK) using an ECL assay kit (Pierce; Thermo Fisher Scientific, Inc.). Western blotting results were analyzed by ImageJ software 1.8.0 (National Institutes of Health, Bethesda, MD, USA).

SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) was used for statistical analyses and all data are presented as the mean ± standard deviation. One-way analysis of variance followed by a Tukey's post-hoc test was used to compare multiple groups and the least significant difference test was performed for pair-wise comparisons. P<0.05 was considered to indicate a statistically significant difference.

In the control, NEC and NEC + berberine groups, the incidence of NEC was 0, 65 and 25%, respectively (Fig. 1). A significant difference in the incidence rate of NEC between the NEC group and NEC + berberine group was observed (Fig. 2). Fig. 2B presents the representative microscopic morphology of the intestinal samples. All rats in grade M or I exhibited bloody diarrhea (data not shown).

In the NEC group, the expression of TLR4 mRNA and protein was significantly and markedly increased, respectively, at 48 h compared with the control group (Fig. 3). However, in the NEC + berberine group, TLR4 mRNA and protein were significantly and markedly suppressed, respectively, compared with the NEC group (Fig. 3).

In the NEC group, NF-κB, iNOS and TNF-α mRNA expression was significantly increased at 48 h compared with the control group (Fig. 4A). However, in the NEC + berberine group, NF-κB, iNOS and TNF-α mRNA expression was significantly decreased compared with the NEC group. Similarly, NF-κB and iNOS protein levels were increased in the NEC group, while these levels were decreased following berberine treatment (Fig. 4B).

In the NEC group, the ileal expression of IL-6 and IL-10 was significantly increased at 48 h compared with the control group (Fig. 5A). However, in the NEC + berberine group, ileal IL-6 and IL-10 were significantly lower compared with the NEC model group. Similar results were observed in serum samples (Fig. 5B).

Compared with the control group, the expression level of MUC2 and SIgA protein was significantly lower in the NEC group at 48 h (Fig. 6). However, in the NEC + berberine group, the expression of MUC2 and SIgA were significantly greater compared with the NEC group.