Primary polyclonal rabbit antibodies against CD3 (ab16669, ̵‌Abcam Plc, Cambridge, UK), CD68 (ab955, ̵‌Abcam Plc, Cambridge, UK), TNF-$\alpha$ (ab6671, ‌Abcam Plc, Cambridge, UK), transforming growth factor-$\beta$1 (TGF-$\beta$1; ab92486, ̵‌Abcam Plc, Cambridge, UK), $\alpha$-smooth muscle actin ($\alpha$-SMA; ab7817, ̵‌Abcam Plc, Cambridge, UK), and JAK1 (ab138005, ‌Abcam Plc, Cambridge, UK) proteins were purchased from Abcam, UK. Additionally, primary polyclonal rabbit antibodies against collagen 1 (14695-1-AP, Proteintech Group, Inc., Chicago, IL, USA) and collagen 3 (227345-1-AP, Proteintech Group, Inc., Chicago, IL, USA) were procured from Proteintech, USA. Phospho-STAT3 (p-STAT3, 381552, Zen Bioscience, Chengdu, China) and total-STAT3 (t-STAT3, 251611, Zen Bioscience, Chengdu, China) were acquired from Zen Bioscience, China. Injectable infliximab (CN-30069V, Cilag AG, Schaffhausen, Switzerland) was sourced from Cilag AG, Switzerland. Furthermore, an improved hematoxylin–eosin (HE) staining kit (G1121, Solarbio Technology Co., Ltd., Shanghai, China) and the modified Movat–Russell pentachrome staining kit (G3700, Solarbio Technology Co., Ltd., Shanghai, China) were purchased from Solarbio, China. In addition, the concentrated SABC-POD (Mouse/Rabbit IgG) kit (SA2010, BOSTER Biological Technology Co., Ltd., Wuhan, China), DyLight 488 conjugated AffiniPure goat anti-mouse IgG (H + L) (BA1126, BOSTER Biological Technology Co., Ltd., Wuhan, China), DyLight 594 conjugated AffiniPure goat anti-rabbit IgG (H + L) (BA1142, BOSTER Biological Technology Co., Ltd., Wuhan, China), ethylenediaminetetraacetic acid (EDTA) antigen retrieval solution (AR0023, BOSTER Biological Technology Co., Ltd., Wuhan, China), 4^′,6-diamidino-2-phenylindole (DAPI) staining solution (AR1176, BOSTER Biological Technology Co., Ltd., Wuhan, China), human TNF-$\alpha$ enzyme-linked immunosorbent assay (ELISA) kit (EK0525, BOSTER Biological Technology Co., Ltd., Wuhan, China), and human TGF-$\beta$1 ELISA kit (EK0513, BOSTER Biological Technology Co., Ltd., Wuhan, China) were obtained from Boster, China. The human collagen 1 ELISA kit (CSB-EL005716HU, CUSABIO BIOTECH CO., Ltd., Wuhan, China) was obtained from CUSABIO, China.

Primary porcine aortic valve interstitial cells (pAVICs) were isolated from porcine aortic valves through a collagenase digestion process, as previously described in the literature (The relevant identification data for the primary cells have been included in the supplementary materials for reference) [24]. In this investigation, pAVICs from passages three to six were employed. The cells were cultured at 37 °C in a humidified environment with 5% CO₂, utilizing Dulbecco’s Modified Eagle’s Medium (DMEM; 11885-084, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; 10270-106, Gibco, Grand Island, NY, USA), 1% penicillin G and streptomycin (15140122, Gibco, Grand Island, NY, USA). The study applied a previously established method for modeling GFs in vitro. Upon achieving approximately 50% to 60% confluency, the cells were allocated into three distinct treatment groups: glucose control group (CTRL), high glucose (HG), and GFs. The CTRL group cells were cultured in a medium containing 5.5 mmol/L glucose, whereas the cells in the HG group were exposed to a medium with 25 mmol/L glucose. The cells in the GF group underwent alternating incubation between 5.5 mmol/L and 25 mmol/L glucose every 12 hours over a 72-hour period. Infliximab is a specific monoclonal antibody that inhibits TNF-$\alpha$, exerting therapeutic effects by suppressing its proinflammatory activity. In vitro data obtained from ELISA in this study confirmed that infliximab treatment effectively blocks TNF-$\alpha$ activation (Supplementary Fig. 2). Concurrently, each group of pAVICs was independently exposed to 10 µg/mL of TNF-$\alpha$ and infliximab to examine the effects of these agents on fibrosis in pAVICs.

Healthy Sprague-Dawley male rats, weighing 180–200 g, were procured from the Jiangsu Institute of Schistosomiasis Control in China. Subsequently, the rats were housed in cages equipped with food and water, situated in an environment maintained under standard conditions: temperature of 22 °C $\pm$ 2 °C, humidity levels of 55% $\pm$ 5%, and a 12-hour light–dark cycle. All experimental procedures were performed in compliance with the protocol approved by the Ethics Committee for Animal Experiments at the Affiliated Wuxi People’s Hospital of Nanjing Medical University (Ethics No. DL2024015). All animal experiments conducted in this study comply with the Regulations on Laboratory Animals issued by the National Science and Technology Commission and the Implementation Rules for the Regulations on Medical Laboratory Animals promulgated by the Ministry of Health. Each cage accommodated 4–5 rats, with the bedding being routinely replaced to ensure a clean and dry environment.

To develop an animal model of diabetes in rats, we administered an intraperitoneal injection of STZ (Sigma-Aldrich Corp., St. Louis, MO, USA) at a dose of 60 mg/kg, as outlined in our previous study [9]. Rats exhibiting blood glucose levels greater than 16.7 mmol/L at one week post-injection were selected for subsequent experimental procedures. The rats were randomly allocated into five distinct groups: the glucose control group (CTRL), the persistent hyperglycemia group (HG), the diabetes with GFs group (GF), the persistent hyperglycemia with infliximab injection group (HG + IFX), and the GF with infliximab injection group (GF + IFX). In the HG + IFX and GF + IFX groups, rats received intraperitoneal injections of infliximab weekly at a dosage of 5 mg/kg for a period of eight weeks. In contrast, other groups were administered physiological saline. The CTRL group was subjected to insulin therapy, receiving long-acting insulin (glargine insulin, 20 IU/kg; Sanofi-Aventis Co., Paris, France) twice daily to ensure stable glycemic control. To induce GFs in diabetic rats, a regimen alternating between 24-hour fasting and 24-hour ad libitum feeding was employed [25]. During the fasting intervals, rats were administered conventional insulin (insulin Aspart, 0.5 IU/kg; Novo Nordisk Corp., Copenhagen, Denmark) to reduce blood glucose levels when they exceeded 5.5 mmol/L. Following an 8-week fasting regimen, the rats were provided with unrestricted access to food for two days, commencing 24 hours before the conclusion of the fasting period, before being euthanized for experimental purposes. For euthanasia, animals were anesthetized with 5% isoflurane, anesthesia was confirmed by tail pinch, and then sacrificed by cervical dislocation. During the experiment, aortic valves were excised, and individual samples were immediately fixed in 4% paraformaldehyde (PFA) for subsequent analysis.

Following the completion of the modeling process, each rat within the respective groups was marked and subsequently administered 10% pentobarbital sodium into the lower abdomen at a dosage of 50 mg/kg of the related body weight to induce anesthesia. Upon achieving adequate anesthesia, the thoracic hair of the rats was carefully removed using a hair clipper to expose the underlying skin. Thereafter, continuous Doppler technology was employed to evaluate aortic valve function across the different rat groups, while left ventricular function was assessed using two-dimensional (2D) ultrasound technology. Specifically, the apical 3-chamber view and the parasternal long-axis view were utilized to evaluate the aortic valve and left ventricular function, respectively. For the comprehensive analysis and acquisition of essential measurement values, the built-in measurement module of the ultrasound diagnostic instrument (Philips IE33, Koninklijke Philips N.V., Amsterdam, Netherlands) was employed. The parameters for cardiac evaluation included left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular internal diameter in systole (LVIDs), left ventricular internal diameter in diastole (LVIDd), aortic root diameter, mean blood flow velocity across the aortic valve (Vmean), peak blood flow velocity across the aortic valve (Vmax), mean transvalvular pressure gradient of the aortic valve (meanPG), and peak transvalvular pressure gradient of the aortic valve (maxPG).

The rat aortic valves were fixed in 4% PFA in phosphate-buffered saline (PBS) at 4 °C overnight. Subsequently, the tissue was dehydrated through a graded ethanol series, treated with xylene, and embedded in paraffin wax. Tissue sections, with a thickness of 6 µm, were prepared using a Leica microtome. HE staining was employed to elucidate the fundamental pathological alterations in the aortic valves of diabetic rats.

The paraffin-embedded aortic valve sections were subjected to anti-detachment treatment, followed by the application of the modified Movat–Russell pentachrome staining protocol, as recommended by the manufacturer. The stained sections were then examined microscopically, revealing distinct coloration: nuclei and elastic fibers appeared black, collagen and reticular fibers were yellow, proteoglycans exhibited a blue-green hue, cellulose-like structures and cellulose structures were dark red, and myocardial smooth muscle was red. Images were captured using a Nikon Eclipse 50i microscope (Nikon, Tokyo, Japan) in conjunction with NIS-Elements F software (NIS-Elements F software Version 4.60, Nikon, Tokyo, Japan). For the morphometric analysis of staining, a minimum of three fields per section were photographed to quantify the intensity and density of the positive signals. Image-Pro Plus 6.0 software (Image-Pro Plus software Version 6.0, Media Cybernetics, Silver Springs, MD, USA) was employed, utilizing the integrated optical density (IOD) parameter and the straw tool to identify positive signals. A selection range was established to filter out impurities, thereby ensuring the reliability of the data. The ratio of IOD to the image area (IOD/area) of the proteoglycan regions was calculated and analyzed for comparison and statistical evaluation among the different rat groups.

For the immunohistochemical analysis of CD3, CD68, TNF-$\alpha$, collagen 1, collagen 3, $\alpha$-SMA, JAK1, and STAT3 expression, our study employed transverse 4% PFA-fixed, paraffin-embedded aortic valve sections (6 µm thick). The procedure commenced with the dewaxing and hydration of the sections, followed by the inhibition of endogenous peroxidase activity using 3% hydrogen peroxide for 30 minutes. Subsequently, the sections were immersed in an EDTA antigen retrieval solution and subjected to microwave heating at high temperature for 5 minutes. This process was allowed to cool to room temperature and was repeated twice. To reduce nonspecific binding, the tissue sections were incubated with 5% goat serum at room temperature for one hour. The primary antibody was applied and incubated at 4 °C overnight. This was followed by a 60-minute incubation with a biotinylated secondary anti-rabbit and anti-mouse IgGs, and detection was performed using 3,3^′-diaminobenzidine (DAB). Hematoxylin staining was applied for counterstaining. The sections were then visualized using a microscope, and the resulting images were quantitatively analyzed using Image ProPlus 6.0 software. The data are presented as the IOD/area.

Cells were lysed using ice-cold RIPA buffer (89900, Pierce Co., Rockford, IL, USA) supplemented with protease and phosphatase inhibitors (04693159001, Roche, Basel, Switzerland), in accordance with the manufacturer’s recommended protocols. Immunoblotting was subsequently employed to analyze collagen 1, TGF-$\beta$1, p-STAT3, t-STAT3, and $\beta$-actin. The lysate was initially subjected to separation via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and subsequently transferred onto a polyvinylidene fluoride (PVDF) membrane (10600121, ̵‌Amersham Biosciences Co., Chicago, IL, USA). To prevent nonspecific binding, the PVDF membrane was then incubated with a 5% skim milk solution at room temperature for one hour. Following this blocking step, the membrane was incubated with the specified primary antibody. After the incubation period, the membrane underwent washing with PBS containing 0.05% Tween 20. Finally, the membrane was treated with a peroxidase-conjugated secondary antibody specific to the primary antibody. Following additional washing steps, the membrane was incubated with enhanced chemiluminescence reagents and subsequently exposed to X-ray film for visualization. The resulting immunoblot bands were quantified using a densitometer in conjunction with ImageJ software (Version 1.8, Scion Corp, Frederick, MD, USA). Density standardization served as a control treatment, while relative folding standardization was applied to $\beta$-actin to ensure the accuracy of the quantification process.

To elucidate the relationship between CD3 and JAK1, as well as CD68 and JAK1 expression, immunofluorescence co-staining was conducted on the aforementioned aortic valve sections. Initially, the paraffin-embedded sections (6 µm thick) underwent dewaxing and hydration processing, followed by the inhibition of endogenous peroxidase activity using 0.3% hydrogen peroxide for 30 minutes. Subsequently, antigen retrieval was achieved by immersing the sections in an EDTA solution and subjecting them to high-temperature microwave treatment for 5 minutes. After cooling to an ambient temperature and repeating the procedure twice, the tissue slices were incubated with 5% goat serum at room temperature for one hour to reduce non-specific binding. Subsequently, primary antibodies from different species were applied and incubated at 4 °C overnight. This was followed by incubation with the corresponding fluorescent secondary antibodies at room temperature in the dark for one hour. Finally, the samples were stained with DAPI, and images were acquired from each slice using a confocal microscope. For the statistical analysis of positively stained cells by each antibody, a minimum of three fields per section were randomly selected. The intensity and density of the positive signals associated with individual cells were quantified using the Image-Pro Plus 6.0 software. The results are expressed as IOD/area.

To assess the efficacy of IFX in inhibiting TNF-$\alpha$ activation and its related downstream signaling pathways, specifically those involving collagen 1 and TGF-$\beta$1, we performed an ELISA on cell supernatants that had been subjected to TNF-$\alpha$ stimulation and IFX treatment. The preparation of samples and standards adhered strictly to the experimental protocols recommended by the manufacturer. Biotin-labeled antibodies were subsequently added, and the reaction was incubated at 37 °C for 60 minutes. Following this, the samples underwent three washes with PBS, after which an avidin–biotin complex (ABC) was introduced, and the reaction was maintained at 37 °C for an additional 30 minutes. The samples were then washed five times with PBS before the addition of the tetramethylbenzidine (TMB) substrate, allowing the reaction to proceed at 37 °C for 15 to 20 minutes. A stop solution was subsequently applied, and the optical density (OD) was measured using a plate reader. A standard curve was generated from the standards to quantify the concentration of the samples.

Data are presented as the mean $\pm$ standard error of the mean (SEM). Statistical analyses were conducted using IBM SPSS Statistics Version 2.0 software (IBM, Armonk, NY, USA). For comparisons between two groups, Gaussian-distributed numerical variables were analyzed using the Student’s t-test, preceded by Levene’s test for equality of variance. For comparisons involving three or more groups, a one-way analysis of variance (ANOVA) was utilized, with subsequent pairwise comparisons performed using Tukey’s honestly significant difference (HSD) test. Non-Gaussian numerical variables were evaluated using the Mann–Whitney non-parametric test. Two-sided probability p-values 0.05 were considered statistically significant.

As depicted above, three distinct groups of rat models were established to examine the effects of GFs on aortic valve function in diabetic rats: CTRL, HG, and GF. The cardiac ultrasound results for these groups after 8 weeks are presented in Fig. 1 (Fig. 1A,F). Compared to both the CTRL and HG groups, the GF group demonstrated significantly elevated Vmax and maxPG values, with these differences reaching statistical significance (p 0.05). Although the Vmax and maxPG values in the HG group were higher than those in the CTRL group, the difference did not attain statistical significance (Fig. 1B,C). Meanwhile, there were no significant differences in the Vmean and meanPG values among the three groups of rats (Fig. 1D,E). Similarly, the inner diameter of the aortic root did not differ significantly among the groups. However, further examination of the impact of blood glucose concentration on left ventricular function revealed that the GF group exhibited significantly reduced LVEF and LVFS compared to the HG group, with these reductions being more pronounced than those observed in the CRTL group (Fig. 1G,H). Furthermore, the LVIDs in the GF group of rats were markedly increased in comparison to both the HG and CTRL groups. Importantly, there was no statistically significant difference in the LVIDd among the three groups (Fig. 1I).

Fig. 1.

Glucose fluctuations adversely affect the function of the aortic valve and left ventricular function in diabetic rat models. (A) The aortic valve functions in diabetic rats from the glucose control (CTRL), high glucose (HG), and glucose fluctuations (GF) groups. (B–E) The peak and mean blood flow velocity across the aortic valve (Vmax and Vmean), as well as the peak and mean transvalvular pressure gradients of the aortic valve (maxPG and meanPG), were measured in diabetic rats from the CTRL, HG, and GF groups (n = 3 per group). (F) The left ventricular function in three cohorts of diabetic rat models. (G–I) The left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and left ventricular internal diameter in systole (LVIDs) were assessed in diabetic rats (n = 5 per group). The data are presented as the mean $\pm$ SEM. Statistical analyses were conducted utilizing one-way ANOVA, followed by post hoc corrections to account for multiple comparisons (Fig. 1B–E, G–I). ^*p 0.05. SEM, standard error of the mean.

The rat aortic valve sections underwent HE staining as well as modified Movat–Russell pentachrome staining to assess the effects of GFs on aortic valve function in vivo. The findings demonstrated heightened expression of proteoglycans in the aortic valve specimens from the HG and GF groups compared to the CTRL group. Furthermore, the aortic valve in the GF group displayed increased thickening relative to the HG group, although the expression level of proteoglycans was not significantly increased (Fig. 2A–C). Immunohistochemical analysis indicated a significant upregulation in collagen 1, collagen 3, and $\alpha$-SMA expression in the rat aortic valve samples from the HG and GF groups compared to the CTRL group (Fig. 3A–C). Notably, the expressions of collagen 1 and collagen 3 were significantly elevated in the GF group relative to the HG group, whereas the $\alpha$-SMA levels did not exhibit significant variations (Fig. 3D–F). These results collectively indicate an enhancement in the fibrotic morphology of the aortic valve in diabetic rats subjected to fluctuations in glucose levels.

Fig. 2.

Inhibition of TNF-$\alpha$ attenuates glucose fluctuations-induced proteoglycan deposition in the aortic valves of diabetic rat models. (A) HE staining was utilized to examine the morphology of the tricuspid aortic valves across five cohorts of diabetic rats. (B,C) Modified Movat–Russell pentachrome staining demonstrated an increased deposition of proteoglycans (indicated by arrows) in the aortic valves of the HG and GF rat cohorts in comparison to the CTRL cohort. However, administration of infliximab reduced proteoglycans (arrows) in both the HG and GF groups (n = 5 per group). The data are presented as the mean $\pm$ SEM. Statistical analyses were conducted utilizing one-way ANOVA, followed by post hoc corrections to account for multiple comparisons (Fig. 2C). ^*p 0.05, scale bar = 100 µm. TNF-$\alpha$, tumor necrosis factor alpha; HE, hematoxylin and eosin; ANOVA, analysis of variance.

Fig. 3.

Inhibition of TNF-$\alpha$ mitigates glucose fluctuations-induced aortic valve fibrosis. (A,B,D,E) Immunohistochemical staining for collagen 1 and 3 (indicated by arrows) demonstrated enhanced collagen deposition in the aortic valves of the HG and GF diabetic rat models. However, treatment with infliximab was observed to mitigate this collagen accumulation. (C,F) Immunohistochemical analysis for $\alpha$-SMA (arrows) revealed an increased fibrotic response in the aortic valves of the diabetic rats in the HG and GF groups; however, infliximab treatment reduced this progression (n = 5 per group). The data are presented as the mean $\pm$ SEM. Statistical analyses were conducted utilizing one-way ANOVA, followed by post hoc corrections to account for multiple comparisons (Fig. 3D–F). ^*p 0.05, scale bar = 50 µm or 100 µm. $\alpha$-SMA, $\alpha$-smooth muscle actin.

Immunohistochemical analysis of the rat aortic valves was employed to quantify the expression levels of inflammatory markers CD3, CD68, and TNF-$\alpha$. Our results indicated a significant upregulation of these markers in both the GF and HG groups compared to the CTRL group (Fig. 4A–C). Importantly, the expression levels of CD68 and TNF-$\alpha$ were markedly higher in the GF group relative to the HG group (Fig. 4D,F). Interestingly, a substantial reduction in the expression of these inflammatory markers was observed after intraperitoneal administration of infliximab (Fig. 4D–F).

Fig. 4.

Inhibition of TNF-$\alpha$ reduced inflammation induced by glucose fluctuations in the aortic valves of diabetic rat models. (A,D) Immunostaining for TNF-$\alpha$ (indicated by arrows) revealed an elevated expression of the TNF-$\alpha$ protein in the aortic valves of the HG group, with an even greater upregulation observed in the GF group. Notably, the inhibition of TNF-$\alpha$ effectively reversed this upregulation in both the HG and GF groups. (B,E) The HG and GF groups exhibited an increased infiltration of CD3-positive T lymphocytes compared to the CTRL group; however, this increase could be mitigated by the inhibition of TNF-$\alpha$. (C,F) The HG group showed increased CD68-positive macrophage infiltration compared to the CTRL group, and the GF group showed even higher levels; however, TNF-$\alpha$ inhibition could reduce this upregulation (n = 5 per group). The data are presented as the mean $\pm$ SEM. Statistical analyses were conducted utilizing one-way ANOVA, followed by post hoc corrections to account for multiple comparisons (Fig. 4D–F). ^*p 0.05, scale bar = 50 µm or 100 µm.

In this study, the expression of fibrosis-associated proteins in pAVICs was evaluated in vitro using Western blot analysis across three groups: CTRL, HG, and GF. The findings indicated that the GF group exhibited elevated levels of TGF-$\beta$1 and collagen 1 compared to the HG group, which, in turn, showed higher levels than the CTRL group. Furthermore, treatment with the TNF-$\alpha$ protein at a concentration of 10 µg/mL resulted in a significant upregulation of TGF-$\beta$1 and collagen 1 expression in all three groups (Fig. 5A,C,E,G). There was a significant reduction in the expression levels of TGF-$\beta$1 and collagen 1 in both the HG and GF groups upon administration of infliximab at a concentration of 10 µg/mL (Fig. 5B,D,F,H). These findings were corroborated by an immunohistochemical analysis, which demonstrated that intraperitoneal administration of infliximab reduced the expression of the fibrotic proteins TGF-$\beta$1 and collagen 1 within the valve tissue of rats in the HG + IFX and GF + IFX groups, compared to the HG and GF groups (Fig. 3A–F).

Fig. 5.

TNF-$\alpha$-mediated inflammation could exacerbate fibrosis in vitro in porcine aortic valve interstitial cells across different glucose levels. (A,B,E,F) In the primary porcine aortic valve interstitial cells (pAVICs) groups, both HG and GF conditions were found to upregulate collagen 1 protein expression. Moreover, the proinflammatory cytokine TNF-$\alpha$ aggravated this upregulation under HG conditions, with an even more pronounced effect observed under GF conditions. Conversely, the inhibition of TNF-$\alpha$ could reverse these upregulations in both the HG and GF groups. (C,D,G,H) TGF-$\beta$1 protein expression increased under both HG and GF conditions, with TNF-$\alpha$ further enhancing this increase, especially under GF conditions. The inhibition of TNF-$\alpha$ reversed these effects in both groups (n = 4 per group). The data are presented as the mean $\pm$ SEM. Statistical analyses were conducted utilizing one-way ANOVA, followed by post hoc corrections to account for multiple comparisons (Fig. 5E–H). ^*p 0.05.

In further in vitro experiments incorporating Western Blot analysis, the expression levels of JAK1 and STAT3 protein were elevated in the GF and HG groups relative to the CTRL group, with the GF group exhibiting the most pronounced upregulation. The application of the TNF-$\alpha$ protein at a concentration of 10 µg/mL further augmented the expression of JAK1 and STAT3 in pVICs within the CTRL + TNF-$\alpha$, HG + TNF-$\alpha$, and GF + TNF-$\alpha$ groups compared to their respective CTRL, HG, and GF counterparts (Fig. 6A,C,E,G). Subsequently, treatment with 10 µg/mL infliximab resulted in a significant reduction in the expression levels of JAK1 and STAT3 (Fig. 6B,D,F,H). Immunohistochemical analysis of the JAK1 and STAT3 protein expressions in the aortic valves of five rat cohorts revealed that the HG and GF groups exhibited significantly elevated expression levels compared to the CTRL group, with the GF group demonstrating the highest expression. Moreover, administration of infliximab via intraperitoneal injection reduced the JAK1 and STAT3 expressions in both the HG + IFX and GF + IFX groups (Fig. 7A–D).

Fig. 6.

TNF-$\alpha$ activated the JAK1/STAT3 pathway in vitro in porcine aortic valve interstitial cells under varying glucose concentrations. (A,B,E,F) In the pAVIC groups, both the HG and GF conditions upregulated the JAK1 protein expression. The presence of TNF-$\alpha$ exacerbated this upregulation under HG conditions, with a more pronounced effect observed under GF conditions. Conversely, the inhibition of TNF-$\alpha$ effectively reversed these upregulations in both the HG and GF groups. (C,D,G,H) In the pAVIC groups, phospho-STAT3 protein expression increased under both the HG and GF conditions, with TNF-$\alpha$ further enhancing this increase, especially in the GF conditions. Inhibiting TNF-$\alpha$ reversed these effects in both the HG and GF groups (n = 4 per group). The data are presented as the mean $\pm$ SEM. Statistical analyses were conducted utilizing one-way ANOVA, followed by post hoc corrections to account for multiple comparisons (Fig. 6E–H). ^*p 0.05. JAK1, Janus kinase 1; STAT3, signal transducer and activator of transcription 3.

Fig. 7.

Inhibition of TNF-$\alpha$ led to the downregulation of the JAK1/STAT3 pathway in vivo in the aortic valves of diabetic rat models. (A,B) Immunohistochemical analysis of JAK1 (indicated by arrows) revealed an upregulation in JAK1 protein expression in the aortic valves of both HG and GF diabetic rat models. Notably, administration of infliximab was found to attenuate this upregulation. (C,D) Immunohistochemistry showed increased p-STAT3 protein expression in the aortic valves of the HG and GF diabetic rats, which was attenuated by the inhibition of TNF-$\alpha$ treatment. n = 5 per group. The data are presented as the mean $\pm$ SEM. Statistical analyses were conducted utilizing one-way ANOVA, followed by post hoc corrections to account for multiple comparisons (Fig. 7B,D). ^*p 0.05, scale bar = 100 µm.

Utilizing immunofluorescence co-staining techniques, this study investigated the spatial and quantitative associations between JAK1 and CD3, as well as JAK1 and CD68. This analysis revealed an enhancement in the co-localization of JAK1 with CD3 and CD68 in both the GF and HG experimental groups (Fig. 8). Notably, the GF group exhibited a greater increase compared to the HG group. Meanwhile, a reduction in the co-staining of JAK1, CD3, and CD68 was observed after infliximab administration via intraperitoneal injection (Fig. 8A,B).

Fig. 8.

Colocalization of CD3, CD68, and JAK1 in the aortic valves of diabetic rat models. (A,B) The coimmunostaining analysis of the HG and GF groups demonstrated the presence of CD3, CD68, and JAK1 proteins, indicating that JAK1 expression is primarily localized in T lymphocytes and macrophages. Notably, this expression pattern could be altered through the inhibition of TNF-$\alpha$. Scale bar = 50 µm. DAPI, 4^′,6-diamidino-2-phenylindole.