Introduction

Milk is well thought-out as a nutritional table and complete food. It helps in offering not only physical, but also mental health. From the ancient time, milk has also been known as both curative and preventive medicine. Different milk components including proteins, vitamins and minerals give nutritional and health benefits that are supported by commercial dairying enterprises and also, different milk-based products are obtainable [1]. In addition to this, the various cow milk products are consumed for the disease preventing, health improving and therapeutic purpose. Furthermore, besides its nutritional value, milk and its products are consumed with the medicines for increasing the dynamic and pharmacokinetic properties of medicines. Milk is also considered as basic and very vital for the regeneration of tissue in Ayurvedic system. The main reason for this property is encrypted and combinational effects of its hormones, vitamins, proteins, minerals and growth factors [2]. Furthermore, the evidences also suggested the effect of different key components of bovine milk and its purified sub fractions or constituents in immune response regulation in various ruminant and non-ruminant species. In addition to this, the role of bovine milk for the immune system in human or overall health is very diverse, wide and indefinable [1]. Surprisingly, the effect of Indigenous cow milk towards specific biochemical immune response and inflammation with specific emphasis on targeted pathways of systemic i.e. blood and localised effect i.e. gut are not yet investigated.

Inflammatory bowel disease (IBD) is a chronic gut inflammation [3]. IBD is rising globally affecting people of all ages and also, the pediatric population. Ulcerative colitis (UC) is mainly considered as disease of inflammatory lesions involving the large intestine counting rectum and then, shifts towards the colon and ultimately, the whole colon. On the other side, Crohn’s disease (CD) can affect any part of the gastrointestinal tract, usually the terminal ileum or the perianal part. Complications of IBD include fluid and electrolyte loss, bleeding, diarrhoea and abdominal pain. Ulcerative colitis induces the activation of Th-2 immune response, whereas Crohn’s disease induces the Th-1 immune response activation [4]. The potential cause of IBD is a dysregulated intestinal microflora that further leads to malfunctioning of the immune response [5]. Cell wall components from bacteria are also used for investigating the different IBD responses. Treatment of peptidoglycan-polysaccharide (PG-PS) from group A Streptococci in the rats induced higher fibrosis compared to human serum albumin that was measured by the enhanced gross abdominal score, procollagen I and III mRNAs and cecal collagen content [6]. Peptidoglycan (PGN) is a thick and exposed cover consisting the bacterial cell wall of Gram-positive bacteria in association with the lipoteichoic acid; however, in Gram-negative bacteria, it is a thin layer that further covered by the coating of thick sheet of lipopolysaccharide (LPS). Amazingly, PGN stimulates an inflammatory immune response. A previous study suggested that intraperitoneal injection of PGN from Gram-positive bacteria stimulated the inflammation and causes the resulting arthritic damage of the joints in animal rat models [7].

In case of IBD patients, the main difficulty is in understanding the pathogenesis that makes it complex to find effective and satisfactory treatment. So, keeping above facts in mind, the present investigation focuses on finding the best milk based treatment from Indigenous (Sahiwal, Tharparkar and Gir), cross-bred (Karan fries and Karan swiss) and exotic cow breed (Holstein friesian) milk that improves the inflammatory conditions induced by peptidoglycan from Staphylococcusaureus. We investigated both systemic and localized gut immune response. Furthermore, the mechanism of milk action is explored by establishing inflammatory conditions on induction of inflammation with peptidoglycan and focusing on inflammatory signaling. This study provides the easily available remedy (milk) to reduce the complications of the IBD patients along with lightening on mechanistic pathway.

Materials and Methods

Materials

All chemicals and reagents are procured from SigmaAldrich, St. Louis, MO, Himedia Laboratories Pvt. Ltd, Mumbai, India and Thermo, USA. Kits were purchased from Koma Biotech, Korea and Cloud Clone Corporation, USA.

Methods

Milk treatment for rats

The animals used in this study were male albino wistar rats (150-200 g body weight) that were obtained from Small Animal House, National Dairy Research Institute, Karnal, Haryana (Institutional Animal Ethics Committee approval letter No. 41-IAEC-18-62). The intestinal inflammatory rat model was developed according to the method of Rahal et al. [8] with some modifications using 10 mg/kg body weight dose of peptidoglycan (PGN) from Staphylococcus aureus by laparotomic procedure. PGN laparotomic injection induced IBD like symptoms. Control rats (Sham) were injected with the sterile solution of normal saline. Milk from different breeds, Sahiwal (SW), Tharparkar (TP), Gir (GIR), Karan Fries (KF), Karan Swiss (KS) and Holstein Friesian (HF) was administered for a period of 28 days after the PGN injection. Milk was administered orally on daily basis in the human equivalent dose following the formula of Shin et al. [8].

Human equivalent dose (mg/kg) = Animal dose (mg/ kg)*Animal Km/Human Km

Different parameters such as weight gain and %stool water content were determined. At the end of study, organ weight index was calculated.

Evaluation of Macrophage phagocytic function

Peritoneal macrophages collected from rats and phagocytic activity was determined according to the method of Dang et al. [9] with little modifications. Briefly, peritoneal fluid containing macrophages was adjusted to 1*10⁶ live cells/ml with the DMEM media containing 10% FBS. About 200μl of cell suspension/well was added in a 96-well, flat-bottomed tissue culture plate. This was followed by the addition of Zymosan (650 μg/ml) and Nitroblue tetrazolium (250 μg/ml) dye in wells. Following this, the plates were incubated in a humidified CO₂ incubator at 37 ^°C with 5% CO₂ for 3 h. Absorbance was read at 570 nm by multiwell scanning spectrophotometer (Epoch^™ Microplate Spectrophotometer, BioTek) after dissolving formazan crystals formed at the bottom of plate in DMSO.

Spleen lymphocytes’ proliferation assay

Spleen lymphocytes’ proliferation assay was done according to the method of Sharma et al. [10] with little modifications. In brief, carefully isolated spleen lymphocytes suspension was adjusted to 1*10 ⁶ cells/ml using the culture media (DMEM) with 10% FBS. About 200μl of this cell suspension was placed in each well of a 96-well, flat-bottom tissue culture plate. The lipopolysaccharide (50μg/mL) was used as a mitogen. The plates were incubated in a humidified CO₂ incubator (5% CO₂) at 37 ^ºC for 24 h. The proliferative potential of cells was estimated by adding 20 μl of MTT solution to each well after culturing lymphocytes for 24 h. The plates were further incubated for 4 h with MTT in a humidified CO₂ incubator at 37 ^ºC. The supernatant was pipetted out. Then, dark blue crystals were dissolved using DMSO. The optical density was read at the test wavelength of 570 nm using ELISA reader (Biotek Elisa reader).

In vivo permeability testing of intestine

In vivo permeability testing was performed using 4000 Da fluorescent dextran-FITC (DX-4000-FITC) according to the method of Cani et al. [11] with little modifications. Blood samples were collected from the eye by retro-orbital plexus puncture and cardiac puncture, respectively after 1 and 4 h. The analysis for DX4000-FITC concentration with a fluorescence spectrophotometer (Epoch^™ Microplate Spectrophotometer, BioTek) at an excitation wavelength of 485 nm and emission wavelength of 535 nm was done.

Measurement of gastric acidity

Change in gastric acidity was determined following the method of Sabiu et al. [12] with little modifications. Gastric acidity was measured by titration with 0.1 N NaOH using Toepfer’s reagent and Phenolphthalein as indicators. The red and pink color was noticed for the measurement of free and total acidity, respectively in gastric content.

Estimation of immunoglobulins, cytokines and inflammatory proteins

The sandwich ELISA assay was used for the measurement of the protein level of interferon gamma (IFN- γ), interleukins (IL10, IL-6), monocyte chemotactic protein (MCP-1), tumor necrosis factor-alpha (TNF-α), immunoglobulin A and G according to the manufacturers’ protocol.

Establishment of in vitro Caco-2 inflammatory model

Cells were plated in 96-well plates at 1 ×10⁴ cells/well. Cells were then treated with various concentrations of peptidoglycan (2.5, 5 and 10μg/ml) for different time periods (2, 4 and 6 h). Cell viability was determined using MTT solution (5 mg/mL PBS).

In vitro simulated gastro-intestinal digestion of milk

A two-step in vitro assay was carried out according to the method of Fotschki et al. [13] with some modifications. Porcine pepsin (800–2,500 units/mg protein), pancreatin (activity, 8×USP specifications) and Bile extract (ox), which possesses cholic acid (approximately 55%), deoxycholic, glycocholic and taurocholic acids were used to simulate human digestion in the stomach and intestine.

Quantitative real time-PCR analysis

Caco-2 cells were cultured in sterile 6-well polystyrene tissue culture plates. Cells were treated with 90% media and 10% PBS in control and PGN group, 10% simulated digested milk from SW and KS breed (DSW and DKS, respectively) for 24 h followed by PGN treatment for 6 h in PGN, PGN+DSW and PGN+DKS group. In addition, cells were pre-treated with DSW and DKS for 24 h followed by media treatment for 6 h in DSW and DKS group. Total RNA was isolated by single step RNA isolation method using 1mL Tri-reagent from 50-100mg intestinal tissue, Caco-2 cells and 1*10 ⁸ peritoneal cells. Reverse transcription was performed using Revert Aid first strand cDNA synthesis kit (Thermo fisher, USA). Relative gene expression was measured by quantitative real-time PCR using the ABI PRISM 7500 Fast detection system (Applied Biosystems) and Maxima SYBR Green (Thermo, USA) master mix. GAPDH was used as an internal control. Primers used are given in Table 1 and 2.

In case of inflammation induced rats, there was significant (p<0.05) enhancement in gut permeability in PGN group as compared to control after 1 h as shown in Figure 2C. Whereas, a significant reduction in gut permeability was found after 1 h in SW, TP, GIR, KF, KS and HF group compared to PGN treated rats (Figure 2C). There was significant (p<0.05) decrease in gut permeability in SW, GIR, KF and KS group as compared to PGN group after 4 h (Figure 2D). However, there was no significant difference in TP and HF milk treated rats as compared to PGN treated rats after 4 h. A significant (p<0.05) enhancement in TP, GIR, KS and HF group and reduction in KF group compared to SW milk treated rats after 4 h was found.

Change in gastric acid secretion

In the present study, we assessed the gastric mucosal protective effect of milk by analyzing free HCl and total acidity. There was a significant (p<0.05) decrease in total acid secretion in SW, TP, GIR, KF, KS and HF group as compared to only PGN treated rats as shown in Figure 2F. But, there was no significant difference in free HCl secretion in milk fed rats compared to PGN group (Figure 2E).

Effect on expression of genes in intestinal tissue

An enhanced expression for the TLR-2 and TLR-4 was found in intestine in PGN group compared to control as shown in Figure 3A and B. The TLR-2 and TLR-4 expression was decreased on feeding with SW, TP, GIR, KF, KS and HF milk in inflammatory conditions as shown in Figure 3. Sodium dependent bile acid transporter (Na-SBT) expression was increased in KF and KS group as compared to PGN group (Figure 4A). An enhancement in ZO-1, Occludin and Claudin expression was found on milk feeding from different breeds in comparison to PGN group as shown in Figure 4. There was no significant difference was found among different groups in Defensin-1 expression (Figure 3 D). In addition to this, approximately seven fold enhancement in GLP-2 expression was observed in GIR and HF group in comparison to control and PGN treated rats, whereas a non-significant difference was observed in SW, TP, KF and KS group as compared to PGN group as shown in Figure 4B. In the present study, about five fold increment in defensin-2 expression was observed in PGN treated rats in comparison to control group. Also, a significant (p<0.05) decrease in defensin-2 was found in SW, TP, KF, KS and HF group compared to PGN group as shown in Figure 3E.