Materials

Sigma Aldrich Company (St. Louis,USA) provided the EGCG. Tween 80 (ADWIC, Egypt). Absolute ethanol (99.5–99.8%) was purchased from Merck. For the immunohistochemical analysis of cell apoptosis and epithelial cell-to-cell adhesion, Dako (Glostrup, Denmark) supplied mouse monoclonal antibodies against human Bcl-2 (JC70A, IgG1) and E-cadherin (1A4, IgG2a).

EGCG nanoemulsion was prepared by a modified oil-in-water (O/W) emulsification method referring to ( Gadkari et al., 2017) using Tween 80 as an emulsifier. To get an O/W nanoemulsion, the prepared ethanolic solution containing EGCG was added drop-wise over a beaker containing 100 ml distilled water and homogenized via probe ultrasonicator (20,000 Hz) for 20 min under an ice bath. Firstly, 100 mg of EGCG was dissolved in 10 ml of ethanol then 0.01 gm of Tween 80 was added over it and mixed very well. The mixture was then stirred to get out the organic solvent (ethanol) with a magnetic stirrer (2,000 rpm) for 4 hours at room temperature. The obtained nanoemulsion was stored in the refrigerator for further use.

Using a high-resolution transmission electron microscope (TEM) from Japan (JOEL JEM-2010) that operated at an accelerating voltage of 200 kV, the morphology and size of the nanoemulsion were examined. The Fourier transform infrared (FTIR) technique was utilised to carry out the spectral analysis of the nanoemulsion. Spectroscopy was performed using a JASCO spectrometer, Japan, and the scan ranged from 4000–400 cm ⁻¹. To measure the surface charge of nanoemulsion and its average size, a zeta analyzer (Zetasizer Nano ZS Malvern) was used to investigate the zeta potential and size distribution.

Morphological analysis

Using a transmission electron microscope, an examination of the size and shape of the EGCG nanoemulsion that had been manufactured revealed the formation of a spherical nanoemulsion that possessed a high degree of homogeneity in shape. Around 130 nanometers was the average size of the particles that were developed ( Figure 1A).

Size distribution and Zeta potential

When determining the colloidal stability and surface charges of the particles, the measurement of the zeta potential is considered. The fact that the Zeta potential of the prepared EGCG nanoemulsion was found to be negative 13.5 mV demonstrates the successful formation of a nanoemulsion ( Figure 1B). The size distribution as well as the polydispersity index (PDI) for the EGCG nanoemulsion as measured by DLS. The measurements detect the size distribution to be 228 nm with PDI =1 ( Figure 1C).

The Fourier transformed infrared spectroscopy (FTIR)

The FTIR spectrum of original EGCG displayed a broad peak in the range of 3500-3300 cm ⁻¹ attributed to the eight OH groups present in its structure. The presence of a fingerprint peak in the EGCG FTIR spectrum at 3357.46 cm ⁻¹ referred to OH groups directly attached to the aromatic ring. Additionally, the EGCG IR spectrum revealed the presence of other bands at 1692 cm ⁻¹, 1616 and 1149 cm ⁻¹ due to vibrations of C=O, C-C (aromatic ring), and C-O bonds (pyranose heterocyclic chain), respectively. Other bands were detected at 1235 cm ^-1 for the O-C=O group and 1035 cm ^-1 for the C-O-C group. On the other hand, the EGCG nanoemulsion showed the peaks corresponding to the OH, C=O, C-C (aromatic ring), and C-O bonds, O-C=O group and C-O-C group at 3367 cm ⁻¹, 1692 cm ⁻¹, 1621 cm ⁻¹, 1140 cm ⁻¹, 1243 cm ⁻¹, and 1028 cm ⁻¹, respectively. The shift observed in the EGCG nanoemulsion peak compared to than the original EGCG confirms its change to the nano form ( Figure 1D).

This study made use of thirty adult male albino rats, each of which weighed between 150 and 180 grammes on average . An earlier method developed by Charan and Biswas was used in the computation of the estimation of the sample size. Five rats in each cage were housed in polycarbonate cages with wire lids at Zagazig University’s Faculty of Medicine. Each cage was numbered, and the rats were kept in a well-ventilated animal house. A normal photoperiod was maintained while the room temperature was kept at 23 degrees Celsius and the humidity was kept at 60 percent. The rats were provided with a dry diet of rat pellets, and they had unrestricted access to water throughout the experiment. After an adjustment period of one week, the rats were randomized into three separate groups of equivalent size: Group I rats were a negative control for other groups. Group II (BoNT) rats were anesthetized by intramuscular injection of xylazine (6 mg/kg) (Sigma-Aldrich, USA) and ketamine (70 mg/kg) (Hospira, USA). The SMG was exposed via surgical incision, and then 5 units of BoNTA (Botox ^®, Allergan Inc., Irvine, CA, USA) diluted in 0.1 ml saline were injected once at the center of the right SMG ⁽²⁴⁾. Group III (BoNT + EGCG nanoemulsion group) rats were anesthetized then the SMG was exposed, and BoNTA was injected at the center of the right SMG, similar to group II. Then, the rats received EGCG nanoemulsions in a once-daily dose of 0.1 ml by oro-gastric intubation ( Koutelidakis et al., 2017). The first oral dose of EGCG nanoemulsions was given on the day of intraglandular BoNTA injection and continued daily for four weeks.

All animal experiments followed ARRIVE’s guidelines and the National Institutes of Health’s lab animal protocol (NIH Publications, revised 1985). The Zagazig University Institutional Animal Care and Use Committee (ZU-IACUC/3/F/368/2022) approved animal care and experimental protocols.

Each rat was euthanized at the end of the experiment using a sodium thiopental overdose (EIPICO, Egypt), which was then followed by cervical dislocation. Five samples from each group were prepared for histological and immunohistochemical analysis after carefully dissecting the appropriate SMGs. The remaining five samples from each group were processed for transmission electron microscopy analysis.

The SMG specimens were first fixed in a formalin solution buffered at 4%, followed by a series of increasing concentrations of ethanol used to dehydrate the glands. Finally, the entire gland was then embedded in paraffin. H&E was used to stain sections that represented the entire sample, which were cut into five micrometer sections. At the Zagazig University Faculty of Medicine, a digital color CCD camera examined and photographed the slides (Olympus, DP73, Tokyo, Japan). A light microscope held it (Olympus BX53, Tokyo, Japan).

Each paraffin block was divided into sections with a thickness of 4 micrometers for immunohistochemical staining. These sections were deparaffinized in xylene and dehydrated in increasing alcohol concentrations. To inhibit internal peroxidase activity, they were submerged in 3% hydrogen peroxide in PBS. Antigen retrieval was carried out in a Panasonic 1380-watt microwave oven for 10 minutes at 2 atm and 120°C. Further incubations with prediluted, ready-to-use primary mouse monoclonal antibodies (anti-Bcl-2, clone MIB-5; Sigma-Aldrich, USA, and anti-E-cadherin; clone HECD-1, Biocare Medical, USA) were used as the primary antibody for 30 min and was incubated in a moist chamber at room temperature (24 h) with a working dilution of 1:50 and 1:100, respectively, followed by the application of secondary antibody (for 15 min), DAB (to produce brown staining), and Meyer’s hematoxylin (for background staining). Following each step mentioned, the samples were put into PBS. According to the manufacturer’s instructions, the positive controls for Bcl-2 and E-cadherin were the kidney and skin, respectively. PBS was used in place of the primary antibody to create the negative control. Brown-colored reaction concentrated in the cytoplasm or nucleus was regarded as a positive reaction. In a blinded analysis carried out by two skilled research associates using a traditional light microscope and Image J software (version 4.10.03, Nikon, Tokyo, Japan), the intensity of the immunostaining was categorized as being negative, weak, moderate, or strong in three fields.

For two hours at room temperature, the 1 mm cubes of SMGs were submerged in a solution of 4 percent paraformaldehyde and 1 percent glutaraldehyde. Before being postfixed in osmium tetroxide at a concentration of 1%, the samples underwent one last rinse in buffer. Following the fixation procedure, the samples were dehydrated in increasing grades of ethanol, immersed in absolute acetone, and then embedded in Epon. At the Mansoura University Electron Microscopy Unit, the RMC-USA ultramicrotome was used to cut the ultrathin slices, then placed on copper grids, stained with uranyl acetate and lead citrate, and viewed with a JEOL JEM-2100 transmission electron microscope (Jeol Ltd, Tokyo, Japan).

Immunohistochemical data were tabulated and statistically analyzed using one-way analysis of variance (ANOVA) to determine group differences Following that, Dunnett’s post-hoc test was utilized to compare the control group to the test group. For the findings to be considered statistically significant, P had to be lower than 0.05. SPSS, version 11.0, was used for the collection and analysis of all statistical data. This software was developed by SPSS Inc. in Chicago, Illinois, in the United States of America.

The architecture of a standard SMG was revealed by the control group. The gland was formed of two main components; the stroma and the parenchyma. Serous acini and various duct types made up the parenchyma. The serous acini were small, rounded, and had narrow lumens. Each acinus is lined with pyramidal cells that have basophilic cytoplasm and large, basal spherical nuclei. Simple cuboidal epithelium with central rounded nuclei and little cytoplasm lining intercalated ducts. Columnar cells with open face nuclei lined the granular convoluted tubules, and the cytoplasm had eosinophilic granules. The striated ducts had high cubical or columnar cells with rounded nuclei and apical brush borders, and basal eosinophilic striations ( Figure 2A). Examination of BoNT group revealed many changes in the SMG. Some acinar cells showed There are many cytoplasmic vacuoles, and there are different types of nuclear shape, size, and number. Other acinar cells had darkly stained cytoplasm and appeared lodged with retained secretory granules. Also, separation between acini was evident. The ducts revealed abnormal architecture, and many ductal cells exhibited cytoplasmic destruction and vacuolization ( Figure 2B). Cells in the ducts and acini showed striking improvement in the BoNT + EGCG nanoemulsion group, in contrast to the BoNT group ( Figure 2C).

Bcl-2 immunohistochemical expression

In the control and BoNT + EGCG nanoemulsion groups, anti-Bcl-2 antibody staining of the cells was found to be weak to moderate. However, the BoNT group’s cells were strongly stained ( Figure 3A-C).

The BoNT group had more Bcl-2-positive immunostained cells than the control and BoNT + EGCG nanoemulsion groups, according to the statistical analysis ( Figure 4 & Table 1).

E-cadherin immunohistochemical expression

Examination of E-cadherin immunoreactivity revealed moderate staining of cells in the control group, weak staining of cells in the BoNT group, and strong staining of cells in the BoNT + EGCG nanoemulsion group ( Figure 5A-C).

When compared to the control and BoNT groups, the E-cadherin positively immunostained cells of the BoNT + EGCG nanoemulsion group showed a significant increase, according to the statistical analysis of the data. The control group had more E-cadherin-positive immunostained cells than the BoNT group ( Figure 6 & Table 2).

The SMG’s normal ultrastructure was visible in the control group. Pyramidal cells with basal nuclei lined the acinar cells. RER parallel arrays were present in well-developed arrays at the base of the cells ( Figure 7A). The epithelium lining the striated duct was simple and columnar in shape, characterized by round euchromatic nuclei, infolded basal membranes, and vertically arranged mitochondria ( Figure 7B). The BoNT group SMG showed many degenerative changes. Acinar cells had many abnormally shaped secretory granules and RER ( Figure 7C). The striated duct showed a loss of normal architecture, and the ductal cells exhibited cytoplasmic rarefaction, dispersed RER, and an abnormal distribution of abnormally configured mitochondria ( Figure 7D). The ultrastructural findings in the BoNT + EGCG nanoemulsion group revealed marked improvement in the SMG compared to the BoNT group and showed nearly normal acini and striated ducts ( Figure 7E & F).

Ahmed Abdelaziz Mohamed Essa: Conceptualization, Data Curation, Resources, Writing – Original Draft Preparation, Writing – Review & Editing

Omneya Mohamed Wahba: Data Curation, Formal Analysis, Investigation, Methodology

Nahla Gamaeldin El-Helbawy: Data Curation, Methodology, Resources, Supervision, Visualization, Writing – Review & Editing

Ola M. El-Borady: Data Curation, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Doaa Ahmed Labah: Data Curation, Formal Analysis, Investigation, Methodology, Writing – Review & Editing