Medium for Group A streptococci (GAS, cat. no. ATCC19615; American Type Culture Collection, Mansas, VA, USA): brain heart infusion (Cat. no. HCM019; Guangdong Huankai Microbial Sci. & Tech4. Co., Ltd., Guangdong, China), the condition for culturing: 24 h at 37 °C. After that, normal saline (NS) was used to wash the cultured GAS. Following inactivation with 10% neutral formalin for 12 h and washing with NS, the GAS were resuspended and the density adjusted to 4.0 $\times$ 10${}^{11}$ colonyforming unit (CFU)/mL in NS as the antigen.

We randomly divided 12 Lewis rats (Cat. no. 113; 150–180 g, eight-week-old, female; Beijing Vital River Animal Technology Co., Ltd., Beijing, China) into 2 groups: the control group (n = 6) and the RHD group (n = 6). Animal experiments conducted in a specific pathogen-free animal laboratory, which belongs to the Animal Experiment Center of Guangxi Medical University. Comfortable living conditions for the rats were guaranteed. The temperature was controlled at 23 °C, with a daily fluctuation of 2 °C, and a 12 h light/dark cycle system. The cages used did not restrict the movement of the rats, and the cages were filled with convenient food and water. The bottom of the cage was covered with soft bedding to protect the feet of rats, as injection of complete Freund’s adjuvant (Cat. no. F5881; CFA, Sigma‒Aldrich, Merck KGaA, Shanghai, China) into the footpad was necessary to establish the RHD model [15, 36]. All animals had 5 days to familiarize themselves with the laboratory environment. All animal experimental procedures were conducted in accordance with the ethical guidelines for the care and use of laboratory animals and were approved by The Animal Care & Welfare Committee of Guangxi Medical University (Approval No. 202209003).

The classical method was used to establish the RHD rat model [14, 37, 38, 39, 40] over a period of 9 weeks. At the beginning of the experiment, rats in the RHD group were treated with unilateral rear footpad injections of a 1:1 (v/v) mixed solution of 0.1 mL of antigen and CFA. After 4 weeks, a 0.5 mL mix of the antigen and CFA solution (1:1, v/v) was injected subcutaneously into the abdomen. After this, the injection solution was changed to 0.5 mL of antigen only, and subcutaneous injections in the abdomen were performed weekly for 4 weeks. The injection protocol for rats in the control group was the same as for those in the RHD group, except that the injection solution contained NS only (the flowchart is shown in Fig. 1A).

Fig. 1.

Increased degree of inflammatory infiltration and fibrosis in RHD group. (A) Flow chart for the establishment of a rat model of RHD. (B) H&E staining of the heart valves of rats. Extensive infiltration of inflammatory cells was observed in the valves of the RHD group compared to the control group; magnification, 400$\times$; scale bar: 50 µm; arrows point to inflammatory cells. (C) Sirius red staining of the heart valves of rats. Increased fibrosis was observed in the valves of RHD rats compared to control rats; magnification, 400$\times$; scale bar: 50 µm; arrows point to COL3. (D) The COL3/1 value in the RHD group was significantly greater than in the control group. (E) RT-qPCR results for fibrosis markers (FSP1, COL1A1 and COL3A1). The expression of FSP1, COL1A1 and COL3A1 in the RHD group was significantly higher than in the control group. (F,G) WB analysis of FSP1 and COL3A1. The protein expression of FSP1 and COL3A1 in the RHD group was significantly greater than in the control group. The data are presented as the mean $\pm$ standard deviation (SD); *p 0.05 vs. the control group. H&E, haematoxylin and eosin staining; COL3, collagen fibre type 3; COL1, collagen fibre type 1; FSP1, fibroblast-specific protein 1; COL1A1, collagen type Ⅰ $\alpha$1 chain; COL3A1, collagen type Ⅲ $\alpha$1 chain; RHD, rheumatic heart disease; RTq-PCR, reverse transcription-quantitative Polymerase chain reaction; WB, western blot.

Following the above treatments, we took approximately 2.5 mL of blood from the retinal vein of rats with isoflurane anaesthesia. All rats were then sacrificed by intraperitoneal injection of sodium pentobarbital (150 mg/kg). The absence of a heartbeat or breathing for more than 5 minutes was considered to indicate death of the animal. If the rats had lost more than 15% of their body weight, and their ability to eat and drink also decreased during the experiment, this was considered the humane endpoint. After collection, heart samples were quickly frozen at –80 °C for follow-up tests. In subsequent experiments, a portion of the heart valve were isolated, then mRNA and protein were extracted from valve tissue for reverse transcription-quantitative Polymerase chain reaction (RT-qPCR) and western blot (WB) analysis [7, 14, 41, 42].

A co-culture method of M1 macrophages grown with ECs was used to simulate the adhesion of M1 macrophages to the endothelium. It was also used to observe whether this cell combination could trigger downstream cascade reactions that increased endothelial permeability. Human monocytic (THP-1) cells (Cat. no. CL-0233; Procell, Wuhan, China) were cultured in THP-1 medium (Cat. no. CM-0233; Procell, Wuhan, China). Human umbilical vein ECs (Cat. no. PRI-H-00023; ZQXZBIO, Shanghai, China) were cultured in EC medium (Cat. no. 1001; ScienCell, San Diego, CA, USA). All cells were cultured in a moist cell incubator at 37 °C and in a 5% CO${}_2$ atmosphere. THP-1 cells were induced to differentiate into macrophages [M0; induced by phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) for 48 h] and then into M1 macrophages [M1; induced from M0 by IFN-$\gamma$ (20 ng/mL) and LPS (100 ng/mL) for 48 h]. M0 or M1 cells were then co-cultured (direct contact) with ECs for 24 h to establish the M0+EC and M1+EC groups. The medium used for co-cultivation was a 1:1 mixture of THP-1 medium and EC medium. All cell lines have passed STR profiling validation and tested negative for mycoplasma, and were cultured in a humidified cell incubator at 37 °C and 5% CO${}_2$.

RT-qPCR was used to quantify the mRNAs for: M1 macrophage biomarkers [inducible nitric oxide synthase (iNOS), CD86, TNF-$\alpha$]; M2 macrophage biomarkers [Arg1 and TGF-$\beta$]; VLA4, VCAM-1, and RAC1; fibroblast-specific protein 1 (FSP1, a fibrosis molecular marker); collagen type I $\alpha$1 chain (COL1A1, a fibrosis molecular marker); and collagen type III $\alpha$1 chain (COL3A1, a fibrosis molecular marker). Table 1 presents all the primers used in this study. RNA was first extracted from samples with AxyPrep reagent (Cat. no. AP-MN-MS-RNA; Axygen, Inc., Hangzhou, China). After that, we used a NanoDrop™ One spectrophotometer (Cat. no. 701-058111; NanoDrop Technologies; Thermo, Inc., Waltham, MA, USA) to measure the concentration of RNA. We then used a cDNA synthesis kit (Cat. no. 11141ES60; Yeasen, Inc., Shanghai, China) to reverse transcribe the total RNA into cDNA, the condition was 25 °C for 5 min, 55 °C for 15 min and 85 °C for 5 min. A CFX96 real-time system (CFX96 TOUCH; Bio-Rad, Inc., Shanghai, China) and qPCR SYBR Green Master Mix (Cat no. 11203ES03; Yeasen, Inc., Shanghai, China) were then utilized to perform RT‒qPCR on the cDNA (95 °C, 5 min; 40 cycles of 95 °C for 10 sec; 55 °C, 20 sec; 72 °C, 20 sec). All procedures were carried out according to the manufacturer’s protocol, and the expression level of each mRNA was expressed as the fold-difference between the mRNA and the internal reference $\beta$-actin using the 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Cq}}$ method [43].

Primary antibodies used for the detection of proteins by WB analysis were: CD86-antibody (1:1000 dilution; cat. no. 13395-1-AP; ProteinTech Group, Inc., Wuhan, China), TNF-$\alpha$-antibody (1:1000; cat. no. 26405-1-AP; ProteinTech Group, Inc., Wuhan, China), Arg1-antibody (1:5000; cat. no. 16001-1-AP; ProteinTech Group, Inc., Wuhan, China), TGF-$\beta$-antibody (1:5000; cat. no. 21898-1-AP; ProteinTech Group, Inc., Wuhan, China), VLA4-antibody (1:2000; cat. no. 19676-1-AP; ProteinTech Group, Inc., Wuhan, China), VCAM-1-antibody (1:2000; cat. no. DF6082; Affinity Biosciences, Jiangsu, China), RAC1-antibody (1:1000; cat. no. 24072-1-AP; ProteinTech Group, Inc., Wuhan, China), p-PYK2-antibody (1:2000; cat. no. AF7383; Affinity Biosciences, Jiangsu, China), p-VE-cad-antibody (1:2000; cat. no. AF3265; Affinity Biosciences, Jiangsu, China), FSP1-antibody (1:5000; cat. no. 16105-1-AP; ProteinTech Group, Inc., Wuhan, China), COL3A1-antibody (1:1000; cat. no. 22734-1-AP; ProteinTech Group, Inc., Wuhan, China), $\beta$-tubulin-antibody (1:1000; cat. no. 10068-1-AP; ProteinTech Group, Inc., Wuhan, China) and GAPDH-antibody (1:3000; cat. no. 10494-1-AP; ProteinTech Group, Inc., Wuhan, China). Each of the above antibodies can react with both rat and human samples. RIPA lysis buffer (Sangon Biotech Co., Ltd.) was used to extract protein from each sample, followed by measurement of the concentration using a bicinchoninic acid (BCA) protein assay kit (Cat. no. C503061; Sangon Biotech Co., Ltd., Shanghai, China). Next, equal amounts of protein (30 µg) from each sample were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 60 V for 30 min, and then 120 V for 60 min. After separation, the proteins were transferred to 0.22 µm polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Darmstadt, Germany) at 400 mA for 30 min. Membranes were then blocked for 1 h at room temperature with a 3% BSA blocking solution (Sangon Biotech Co., Ltd.). Finally, the membrane was incubated with primary antibody overnight at 4 °C, washed, and subsequently incubated at room temperature for 1 h with the HRP-conjugated secondary antibody (1:5000; cat. no. S0001; Affinity Biosciences). Finally, the protein bands were visualized with a touch imager system (E-blot, Inc., Shanghai, China), and the amount of protein quantified with ImageJ software (1.51j, National Institute of Health, Bethesda, MD, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or $\beta$-tubulin were used for normalizing each protein.

Haematoxylin and eosin (H&E) staining and Sirius red staining were employed for histological studies. The test object was a 5 µm paraffin section embedded with valve tissue. The valve tissue was fixed with 4% paraformaldehyde at 4 °C for 24 h, dehydrated in alcohol, and then embedded in molten paraffin wax (58–60 °C). H&E staining was performed on paraffin sections at room temperature, with haematoxylin staining for 4–10 min, and eosin staining for 0.5–2 min. Paraffin sections were stained with Sirius red solution for 1 h. A BX43 light microscope (Olympus Corporation, Tokyo, Japan) was used to record images.

After co-culturing M1 macrophages and ECs on a 6-well plate for 24 h, the cells were washed three times with PBS and then fixed with 4% paraformaldehyde for 10 min at 4 °C. They were then washed three times with PBS, permeabilized with 0.5% Triton X-100 for 20 min, and washed again three times with PBS. The cells were then treated for 30 min with blocking solution (Cat no. P0260; Beyotime, Shanghai, China) and washed. Next, they were incubated overnight at 4 °C with the following primary antibodies: VLA4-antibody (1:100; cat. No. 19676-1-AP; ProteinTech Group, Inc., Wuhan, China), VCAM1-antibody (1:200; cat. No. DF6082; Affinity Biosciences, Jiangsu, China), RAC1-antibody (1:200; cat. no. 24072-1-AP; ProteinTech Group, Inc., Wuhan, China), p-PYK2-antibody (1:200; cat. no. 11216; Signalway Antibody LLC, Greenbelt, MD, USA), and p-VE-cad-antibody (1:200; cat. No. AF3265; Affinity Biosciences, Jiangsu, China). The next day, the samples were washed three times with PBST before incubation with fluorescent secondary antibody (Abcam, Cambridge Science Park, UK) for 1 hour at room temperature in the dark. After washing three times with PBS, 4${}^{\prime }$,6-diamidino-2-phenylindole (DAPI) was added and the samples were incubated in the dark for 10 min. The samples were then washed three times with PBS and viewed with a fluorescence microscope (Cat. no. AMAFD1000; Life Technologies Corporation, Waltham, MA, USA).

The primary antibodies used for immunohistochemistry were as follows: CD86 (1:80; cat. no. 13395-1-AP; ProteinTech Group, Inc., Wuhan, China), TNF-$\alpha$ (1:80; cat. no. 26405-1-AP; ProteinTech Group, Inc., Wuhan, China), VLA4 (1:80; cat. no. 19676-1-AP; ProteinTech Group, Inc., Wuhan, China), VCAM-1 (1:80; cat. no. DF6082; Affinity Biosciences, Jiangsu, China), p-PYK2 (1:80; cat. no. AF7383; Affinity Biosciences, Jiangsu, China), p-VE-cad (1:80; cat. no. AF3265; Affinity Biosciences, Jiangsu, China), interleukin (IL)-6 (1:65; cat. no. GB11117; Servicebio, Wuhan, China), and IL-17 (1:90; cat. no. GB11110-1; Servicebio, Wuhan, China). The secondary antibodies used were anti-rabbit horseradish peroxidase (HRP)-conjugated (1:200; cat. no. GB23303; Servicebio, Wuhan, China). Before incubating the primary antibody, we first sliced the formalin-coated paraffin blocks into 5 µm paraffin sections, blocked the sections with BSA for one hour at room temperature, and then inactivated the peroxide with the hydrogen peroxide enzyme. Next, the samples were incubated with primary antibody for 12 h at 4 °C, followed by incubation at room temperature for 30 min with secondary antibody. After completion of the antibody incubation, diaminobenzidine (DAB) was used for colour development. Finally, a BX43 light microscope (Olympus Corporation) was used to capture images, with brownish-yellow staining indicative of positive cells. We also used a method described in a previous report by Friedrichs et al. [44] to quantify the immunohistochemistry results. Briefly, the immunohistochemical score (IHS) was calculated as the staining intensity (negative = 0 points; weak = 1 point; moderate = 2 points; strong = 3 points) $\times$ percentage of positive staining (negative = 0 points; 10% =1 point; 11–50% = 2 points; 51–80% = 3 points; $>$80% = 4 points).

Permeability experiments were used to measure the effect of M1 macrophages on endothelial permeability [45]. ECs were first randomly divided into two groups (M0+EC group and M1+EC group) and the cell concentration adjusted to 1 $\times$ 10${}^5$ cells/mL. The ECs were used to inoculate a transwell chamber, which was then inserted into a 24-well cell culture plate and 1 mL of medium added to the lower chamber. This was subsequently placed for 24 h in a 5% CO${}_2$ incubator at 37 °C in order for the cells to reach confluence. Subsequently, M0 cells and M1 macrophages induced by THP-1 cells were added to the endothelial monolayers of the M0+EC group and the M1+EC group, respectively, and cultured for 24 h. The transwell chamber was then removed, washed twice with PBS, and the cells placed into a new 24-well plate. PBS was then added to the lower chamber and 200 µL of PBS containing FITC-labelled dextran (1 mg/mL; Sigma) was added to the upper chamber and incubated at 37 °C for 1 h. Finally, we collected PBS from the lower chamber and analysed it using a multifunctional enzyme-linked immunosorbent assay (SuPerMax 3100; Flash Co., Shanghai, China), 494 nm for excitation wavelength and 520 nm for emission wavelength.

The serum levels of IL-6 and IL-17 in rats were measured using ELISA kits (cat. no. E04640r; E07451r; Cusabio Technology LLC, Wuhan, China) according to the manufacturer’s protocols. The absorbance at 450 nm in each well was measured using a microplate reader (Multiskan; Thermo Inc., Waltham, MA, USA).

Statistical analysis of data was performed with SPSS software 17.0 (SPSS, Inc., Chicago, IL, USA), and the results presented as the mean $\pm$ standard deviation (SD). The statistical method used was Student’s t test (comparing differences between two groups), and the criterion for defining a significant difference was p 0.05.

The H&E staining results indicated a greater degree of inflammatory cell infiltration in the heart valves of RHD rats compared to the control group (Fig. 1B). Sirius red staining also indicated a significantly higher level of fibrosis in the heart valves of RHD rats (Fig. 1C). According to the results of Sirius red staining, loosely arranged green fibres represent type 3 collagen (COL3) fibres, closely packed yellow and red fibres represent type 1 collagen (COL1) fibres, and the ratio of COL3 to COL1 increases with the degree of tissue fibrosis. The COL3/COL1 (COL3/1) ratio can therefore be used to represent the degree of valvular fibrosis [46]. The Sirius red staining also showed the COL3/1 ratio was significantly greater in the RHD group than in the control group (p 0.05; Fig. 1D). Moreover, the expression of various fibrosis markers (FSP1, COL1A1 and COL3A1) in heart valves was also significantly higher than in the control group (p 0.05; Fig. 1E–G).

Immunohistochemistry and ELISA also revealed significantly higher levels of IL-6 and IL-17 in the heart valves and serum of RHD rats compared to the control group (p 0.05; Fig. 2A–C).

Fig. 2.

IL-6 and IL-17 expression in the heart valves and serum. (A,B) Immunohistochemistry results for IL-6 and IL-17. Scale bar: 50 μm. (C) Enzyme-Linked Immunosorbent Assay (ELISA) results for IL-6 and IL-17. (A), (B) and (C) showed that IL-6 and IL-17 expression in the heart valves and serum of RHD rats was significantly greater than in the control group. The data are presented as the mean $\pm$ SD; *p 0.05 vs. the control group. IL, interleukin; RHD, rheumatic heart disease.

The results of RT-qPCR, WB and immunohistochemistry showed that the expression of M1 macrophage biomarkers (iNOS, CD86, and TNF-$\alpha$) in the heart valves of RHD rats was significantly higher than in the control group. Moreover, expression of the M1 biomarkers was significantly higher than that of the M2 macrophage biomarkers (Arg1 and TGF-$\beta$). These results suggested that M1 macrophages may play a more important role in the pathogenesis of RHD than M2 macrophages (p 0.05; Fig. 3A–E).

Fig. 3.

Differences in the expression of M1/M2 macrophage biomarkers between control group and RHD group. (A,B) WB analysis of CD86, TNF-$\alpha$, Arg1 and TGF-$\beta$. Protein expression levels of the M1 macrophage biomarkers CD86 and TNF-$\alpha$ were significantly higher in the RHD group than in the control group. Moreover, the M1 macrophage biomarker levels were significantly higher than the M2 macrophage biomarkers Arg1 and TGF-$\beta$. (C) RT‒qPCR results for iNOS, CD86, TNF-$\alpha$, Arg1 and TGF-$\beta$. The mRNA expression levels of iNOS, CD86 and TNF-$\alpha$ were significantly higher in the RHD group than in the control group, and were also significantly higher than the levels of the M2 macrophage biomarkers Arg1 and TGF-$\beta$. (D,E) Immunohistochemical results for CD86 and TNF-$\alpha$ in heart valves. Magnification, 400$\times$; scale bar: 50 µm; arrows point to positive staining. The expression levels of CD86 and TNF-$\alpha$ were significantly higher in the RHD group than in the control group. The data are presented as the mean $\pm$ SD; *p 0.05 vs. the control group. iNOS, inducible nitric oxide synthase; RHD, rheumatic heart disease.

The results obtained with RT-qPCR, WB and immunohistochemistry showed that the RHD group had significantly higher expression levels of VLA4, VCAM-1, RAC1, p-PYK2, and p-VE-cad than the control group. The increased expression of p-VE-cad suggested a possible increase in endothelial permeability, which may be related to the migration of inflammatory cells across the endothelium (p 0.05; Fig. 4A–E).

Fig. 4.

Differences in the expression of VLA4/VCAM-1/RAC1/p-PYK2/p-VE-cad between control group and RHD group. (A,B) WB analysis of VLA4, VCAM-1, RAC1, p-PYK2 and p-VE-cad. The protein expression levels of VLA4, VCAM-1, RAC1, p-PYK2 and p-VE-cad were significantly higher in the RHD group than in the control group. (C) RT‒qPCR results for VLA4, VCAM-1 and RAC1. The mRNA expression levels of VLA4, VCAM-1 and RAC1 were significantly higher in the RHD group than in the control group. (D,E) Immunohistochemical results for VLA4, VCAM-1, p-PYK2 and p-VE-cad in heart valves. Magnification, 400$\times$; scale bar: 50 µm; arrows point to positive staining. The expression levels of VLA4, VCAM-1, p-PYK2 and p-VE-cad were significantly higher in the RHD group than in the control group. The data are presented as the mean $\pm$ SD; *p 0.05 vs. the control group. VLA4, very late antigen4; VCAM-1, vascular cell adhesion molecule-1; RAC1, RAS-related C3 botulinum substrate 1; p-PYK2, phosphorylated proline-rich tyrosine kinase 2; p-VE-cad, phosphorylated VE cadherin; RHD, rheumatic heart disease.

Results obtained with RT-qPCR, WB and immunofluorescence revealed that the expression of M1 macrophage markers (CD86, TNF-$\alpha$), VLA4, VCAM-1, RAC1, p-PYK2, and p-VE-cad were significantly higher in the M1+EC group than in the M0+EC group. These results showed that direct contact between M1 macrophages and ECs can increase the expression levels of VLA4, VCAM-1, RAC1, p-PYK2, and p-VE-cad. The increased expression of p-VE-cad, a key indicator of endothelial permeability, suggested that M1 macrophages may enhance endothelial permeability (p 0.05; Fig. 5A–D).

Fig. 5.

Differences in the expression of VLA4/VCAM-1/RAC1/p-PYK2/p-VE-cad and endothelial permeability between M0+EC group and M1+EC group. (A,B) WB results for VLA4, VCAM-1, RAC1, p-PYK2 and p-VE-cad. The protein expression of VLA4, VCAM-1, RAC1, p-PYK2 and p-VE-cad was significantly higher in the M1+EC group than in the M0+EC group. (C) RT‒qPCR results for CD86, TNF-$\alpha$, VLA4, VCAM-1 and RAC1. The mRNA expression levels of CD86, TNF-$\alpha$, VLA4, VCAM-1 and RAC1 were significantly higher in the M1+EC group than in the M0+EC group. (D) Immunofluorescence results for VLA4, VCAM-1, RAC1, p-PYK2 and p-VE-cad. The expression levels of VLA4, VCAM-1, RAC1, p-PYK2 and p-VE-cad were significantly higher in the M1+EC group than in the M0+EC group. Scale bar: 200 μm. (E) Results of the endothelial permeability assay for the M0+EC and M1+EC groups. The endothelial permeability of the M1+EC group was significantly greater than that of the M0+EC group. The data are presented as the mean $\pm$ SD; *p 0.05 vs. the control group. VLA4, very late antigen4; VCAM-1, vascular cell adhesion molecule-1; RAC1, RAS-related C3 botulinum substrate 1; p-PYK2, phosphorylated proline-rich tyrosine kinase 2; p-VE-cad, phosphorylated VE cadherin; RHD, rheumatic heart disease.

To further verify whether M1 macrophages enhance endothelial permeability, we conducted a permeability assay. Endothelial permeability in the M1+EC group was found to be significantly greater than in the M0+EC group. These findings suggested that M1 macrophages may increase endothelial permeability, thereby promoting the migration of inflammatory cells across the endothelium and into target tissue (p 0.05; Fig. 5E).