Phlorizin Regulates Synovial Hyperplasia and Inflammation in Rats With Rheumatoid Arthritis by Regulating the mTOR Pathway

Materials and Methods

Animals. A total of 30 healthy Wistar rats (male; aged 7-8 weeks; weight: 120-150 g) purchased from Shanghai SLAC Laboratory Animal Co., Ltd., (Shanghai, PR China) were used for this study and housed under standard conditions (55±5% humidity and 25±2°C) with 12-h light and dark cycles. All the animals were provided unrestricted access to food and hydration. The Second People’s Hospital of Nanyang City in Nanyang, China, approved experimental protocols on these animals (reg no. 2023/RA/758493).

Arthritis model. The animals were divided into two distinct groups: A control group (n=15) and a second group (n=15) which received bovine type II collagen (CII) (Sigma-Aldrich, Shanghai, PR China), to induce RA. CII was prepared at 2 mg/ml by dissolving it in 0.05 M acetic acid and mixing it at 4°C. Further, this solution was then mixed at a ratio of 1:1 with Freund’s incomplete adjuvant to prepare a 1 mg/ml solution. Animals were injected subcutaneously in the tail with this solution on the first day (300 μl) and day (200 μl), whereas the control group received injections with saline (200 μl).

Treatment strategy. The CII-treated animals were further split into three groups: the RA control group (n=5) received saline (200 μl); two RA groups (n=5 each), received phlorizin (Shaanxi Lonier Herb Bio-Technology Co., Ltd, PR China) at 60 or 120 mg/kg per os for 3 weeks from the 21^st day after the induction of RA (i.e., total period of the protocol was 7 weeks).

Assessment of clinical signs of inflammation. The rats were weighed and monitored once a week for clinical signs of arthritis from immunization up to 50 days (10).

Estimation of arthritis score. The arthritis severity was assessed by independent researchers was scored 0-4 for each hindlimb as follows: No swelling or redness: 0; redness in the foot or swelling of the ankle: 1; redness and inflammation from midfoot to ankle: 2; entire foot swollen: 3; loss of mobility associated with a swollen foot: 4. The arthritic index was calculated as the total score of the four limbs of each animal (maximum total score=16). An arthritis index greater than 6 points is judged to indicate the successful induction of RA in the model (11). As the arthritis score only provides a subjective quantification of inflammation, it was coupled with the measurement of hind paw diameter using a digital caliper (Fischer Darex, Le Chambon-Feugerolles, France). The values were expressed as the mean of the two hind paw diameters. When indicated, values for individual hind paw diameters are presented.

The mean arthritis index of each group of mice was calculated by dividing the total arthritis score for all mice in a group by the number of mice in the group.

Determination of thymus and spleen index. At 50 days following the induction of RA, the rats were sacrificed. The thymus and spleen were resected from each animal and each of these organs was weighed. Thymus and spleen indices were estimated using the formula given in (12):

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Determination of serum interleukin-1 β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), nuclear factor kappa B (NF-κB), MMP-1 and MMP-3. After rats were sacrificed, TNF-α, IL-1β, IL-6, NFκB, MMP-1, and MMP-3 levels were assessed in the serum of the CII-treated rats by enzyme-linked immunosorbent assay as per the manufacturer’s kit instructions (ThermoFisher, Beijing, PR China) (10).

Histopathological study. Histopathological analysis was performed on sections of paw joints. Ankle joints were analyzed by removing the skin and muscle from the right limb and fixing the tissue in a polyoxymethylene solution (4%) for 1 week at 4°C after isolation. The tissue was subjected to decalcification using a 14% ethylenediaminetetraacetic acid (EDTA) solution, followed by fixation at a temperature of 4°C for 35 days. The joint tissue was dehydrated using alcohol and afterward embedded in paraffin. The tissue was then sectioned into slices with a thickness of 5 μm. The tissue sections were stained with hematoxylin and eosin, and a trinocular microscope (BA210 LED Trinocular; Motic, Wetzlar, Germany) was used for the observation of histopathological changes. All the histopathological alterations were noted quantitatively. The slides were analyzed and histopathologically scored as previously described by López-García et al. (13).

In-vitro cell culture. FLSs were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in medium containing 10% fetal bovine serum. The cells were then washed three times using phosphate-buffered saline solution, followed by treatment with trypsin (0.25%) for the purpose of digestion. The digestive process was ended addition of 10% fetal bovine serum. Cells were cultured in an incubator with CO2 at 37°C using Dulbecco’s modified Eagle’s medium/F12 medium.

Determination of cell proliferation. The effect of phlorizin on cellular proliferation induced by TNFα in FLSs was assessed in vitro. Cells (5×10³) were seeded onto a 24-well plate and exposed to several doses of phlorizin (25, 50, 75, and 100 μM) for 2 h, TNFα (10 ng/ml) was then used to stimulate the cells for 2 days. CCK-8 solution (10 μl) was then added to each well and plates were incubated for 2 h at 37°C. The absorbance at 450 nm was finally measured using a microplate analyzer. Assessment of FLS proliferation was observed with 5-ethynyl-2-deoxyuridine (EdU); cells were incubated for 2 days. The cells were then fixed using paraformaldehyde, and staining was conducted using an Apollo^® staining reaction solution. Further, Hoechst 33342 stain was applied to stain the nucleus, and cellular images were taken with a high-resolution imaging system (EVOS XL Cell Imaging System, Invitrogen™ AMEX1000; ThermoFisher Scientific, Shanghai, PR China). This experiment was conducted three times.

Assessment of cellular apoptosis. Cell apoptosis was determined using a commercial fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit (BD Pharmingen, San Diego, CA, USA) with annexin V-allophycocyanin/propidium iodide double staining, according to the manufacturer’s instructions. A flow cytometer was used to determine the total number of apoptotic cells.

Immunofluorescence staining. The estimation of microtubule-associated protein light chain 3 (LC3B) expression in FLSs was performed using immunofluorescence staining and immunofluorescence microscopy. Cells were stimulated for 2 days with 50 ng/ml TNFα (Sigma-Aldrich, and fixed for 30 min with para-formaldehyde (4%). Cells were incubated at 4°C with antibody to LC3B (2 μl; Thermo Fisher Scientific, Shanghai, China), overnight, and later incubated with a secondary antibody. Images were then captured after staining the cells with 4’,6-diamidino-2-phenylindole Finally, a quantitative assessment was conducted on the cells.

Western blot assay. The synovial tissues were obtained from all the animals, and protein was extracted from the tissue. The protein concentration was then determined using a bicinchoninic acid assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8-15%) was used to separate the proteins, which were transferred to a nitrocellulose filter membrane (0.22 μm). The membrane was incubated at 4°C overnight with primary antibodies targeting protein kinase B (AKT), phospho-AKT (p-AKT), mTOR, p-mTOR, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), p-PI3K (ThermoFisher); hypoxia-inducible factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), light chain 3 II (LC3 II), light chain 3 I (LC3 I) and β-actin (Sigma-Aldrich). Following this, the samples were treated for an additional hour with secondary antibodies. An Odyssey Infrared Imaging System, (LI-COR Biosciences, Lincoln, NE, USA) was used to observe the membrane, while ImageJ (National Institutes of Health, Bethesda, MA, USA) tool was deployed for quantitative analysis of the blots relative to standard blots.

Statistical analysis. The data underwent statistical assessment using SPSS software (Version 19.0; IBM, Armonk, NY, USA). The data are reported as the mean±standard deviation. The ratios were compared using chi-square analysis. A one-way analysis of variance (ANOVA) was performed to compare the means of the two groups. Subsequently, the Fisher least significant difference test was conducted to determine significant differences between the groups. The statistical threshold was set at p<0.05.