Sustained Release of Dual p38 Inhibitors via Supramolecular Hydrogels to Enhance Cardiac Repair after MI/R Injury

Sustained Release of Dual p38 Inhibitors via Supramolecular Hydrogels to Enhance Cardiac Repair after MI/R Injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sustained Release of Dual p38 Inhibitors via Supramolecular Hydrogels to Enhance Cardiac Repair after MI/R Injury Di Wang, Aoxue Xu, Haitao Su, Youpei Zhang, Lingling Jiang, Yaguang Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5944861/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Activation of p38 mitogen-activated protein kinase plays an important role in the progression of ventricular muscle inflammation after myocardial ischemia-reperfusion (MI/R). The inhibition of p38 activation in ischemic myocardium can reduce ventricular muscle remodeling post-MI. However, owing to the dynamic change of p38 in ischemic myocardium after MI, the clinical therapeutic effect of p38 inhibitors is insufficient. Herein, we describe the design of a hydrogelator Nap-Phe-Phe-Thr-Gly-Tyr-OH (Nap-TGY) to coassemble the p38 inhibitor SB202190 (SB), a p38 responsive supramolecular hydrogel (Gel Nap-TGY+SB) for local administration and p38 responsive release of SB to efficiently improve the inflammatory microenvironment. Under the overexpression of p38 in ischemic myocardium, Nap-TGY in the hydrogel is phosphorylated to yield hydrophilic Nap-Phe-Phe-Thr(H2PO3)-Gly-Tyr(H2PO3) (Nap-TpGYp), triggering the disassembly of the hydrogel and a responsive release of the inhibitor. Injection of hydrogel into the ischemic myocardium significantly reduces p38 phosphorylation, mitigates inflammation, and enhances angiogenesis. These findings suggest a novel therapeutic strategy for ischemic cardiomyopathy through modulation of the p38 mitogen-activated protein kinase (MAPK) pathway. Myocardial ischemia-reperfusion supramolecular hydrogels p38 mitogen-activated protein kinase phosphorylation dual inhibition. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Timely implementation of interventions aimed at revascularization can effectively reduce perioperative mortality in patients with myocardial infarction (MI). However, myocardial reperfusion may also result in myocardial ischemia-reperfusion (MI/R) injury, leading to cardiac cell death [ 1 ]. MI/R injury encompasses cardiac hypertrophy, myocardial fibrosis, deformation of the heart chamber, as well as reduced diastolic and systolic functions, ultimately resulting in the development of heart failure [ 2 ]. Following MI/R, a sequence of inflammatory cascades ensures within the ischemic myocardium, ultimately resulting in further injury. This process involves inflammatory cells infiltration, particularly of neutrophils and monocytes, and subsequent secretion of chemokines and cytokines [ 3 ]. The activation of toll-like receptors (TLR)-mediated pathways, the complement system, and reactive oxidative species (ROS) generation by pro-inflammatory signals, along with the modulation of mitogen-activated protein kinase (MAPK)-related pathways, expedites the inflammatory response. Inhibiting the TLR4/MyD88/NF-κB pathway or reducing ROS production can mitigate inflammation and myocardial fibrosis [ 4 , 5 ]. Early treatment to reduce inflammation can help prevent negative changes in the heart structure. P38 MAPKs are a family of stress-activated kinases, together with extracellular signal-regulated kinases 1/2 (Erk1/2), c-Jun N-terminal Kinases (JNKs), extracellular signal-regulated kinases (Erk5), which constitute the four branches of the MAPK system [ 6 ]. The activation of this system has been implicated in various pathological stress responses within the body. Moreover, activation of p38 MAPK is linked to various heart conditions, such as dilated cardiomyopathy, heart attacks, and fibrotic cardiomyopathy [ 7 – 9 ]. Recent studies on p38 MAPK have primarily focused on pharmacological approaches and revealed that inhibiting p38 may enhance myocardial contractility and mitigate MI/R injury [ 10 – 12 ]. Traditionally, p38 is believed to be dual phosphorylated by upstream dual-specific MAPK kinases (MAPKKs) such as MAP Kinase Kinase 3 (MKK3) and MAP Kinase Kinase 6 (MKK6) [ 13 ]. P38 MAPK is known to exist in four isoforms (α, β, γ, δ), all of which contain the Thr-Gly-Tyr (TGY) amino acid sequence. Activation of p38 requires dual phosphorylation at Thr180 and Tyr182 sites [ 14 ]. Crystal structure analyses have revealed that the specificity of p38 MAPK is due to the Gly residue in the Thr-Gly-Tyr (TGY) motif, distinguishing it from other MAPK pathway molecules, such as Erk and JNK [ 15 ]. SB202190, a compound that inhibits the p38 MAPK pathway by binding to the ATP-binding site of p38 MAPK and reducing its activity [ 10 , 16 ]. Previous studies have shown that SB202190 improves heart function, decreases heart inflammation, and prevents heart cell death in both organisms and laboratory setting [ 17 – 19 ]. Its specificity makes it a valuable tool for studying cell signaling pathways linked to p38 overexpression, especially in the context of inflammation therapy. SB202190 exhibits strong hydrophobic properties and requires frequent administration in animal studies. Therefore, choosing an appropriate carrier for targeted delivery to the inflammatory site is important for improving its effectiveness. Recent studies have demonstrated that hydrogel can enhance the solubility and absorption rate of insoluble drugs, improve targeting, decrease adverse reactions, and enable more precise release compared with traditional drug carriers [ 20 – 23 ]. To address these inherent challenges, the present study investigates the utilization of a biocompatible hydrogel loaded with SB202190 (SB) for controlled release in the infarcted region, with the aim of modulating inflammation and reducing ischemia-reperfusion injury. Given the structural similarity between the hydrogel and the phosphorylation binding site and the known inhibitory effects of SB on p38 MAPK phosphorylation, this approach holds promise as a potential dual-action therapy for myocardial ischemia. In this study, we first developed a p38 Mark kinase-triggered gel-to-solution transition strategy for local delivery and controlled release of p38 inhibitors in the ischemic myocardium to ameliorate the pro-inflammatory microenvironment. Specifically, we rationally designed a hydrogelator, Nap-Phe-Phe-Thr-Gly-Tyr-OH (Nap-TGY), consisting of i) a commonly used self-assembling motif Nap-Phe-Phe, and ii) a dipeptide, Thr-Gly-Tyr-OH (TGY), for p38-recognized phosphorylation. Supramolecular hydrogel Nap-TGY (Gel Nap-TGY) was easily obtained via a heating-cooling method. The p38 inhibitor SB was co-assembled with Nap-TGY to yield the hydrogel Nap-TGY + SB (Gel Nap-TGY + SB). We propose that, upon p38 activation, Nap-TGY in the hydrogels is efficiently converted to its hydrophilic phosphate, Nap-Phe-Phe-Thr (H2PO3)-Gly-Tyr(H2PO3) (Nap-TpGYp), triggering the disassembly of the hydrogels and the release of SB in a sustainable manner (Fig. 1 a). After the hydrogel is locally injected, p38 responsive release of SB at the ischemic myocardium site is achieved (Fig. 1 b). In turn, the released SB downregulates p38 activity and consequently inhibits its downstream proteins to efficiently improve the inflammatory microenvironment. Results and Discussion Hydrogels preparation and characterization Initially, a solid-phase peptide (SPSS) was utilized to synthesize the hydrogelator Nap-TGY and the control hydrogelator Nap-Phe-Phe-Ala-Gly-Tyr-OH (Nap-AGY) (Scheme S1 and Scheme S2, Supporting Information). The molecular structures of Nap-TGY and Nap-AGY were confirmed by high-resolution mass spectrometry (HR-MS), 1 H nuclear magnetic resonance ( 1 H NMR), and 13 C NMR spectroscopy (Figure S1 -S6). Subsequently, the physical properties of Gel Nap-TGY, Gel Nap-TGY + SB, and Gel Nap-AGY were assessed through a series of experiments. The dynamic strain scanning characteristics of the three hydrogels were examined using a rheometer. At a strain amplitude of 1.0%, the storage modulus (G’) of Gel Nap-TGY and Gel Nap-TGY + SB were notably higher than their loss modulus (G’’) within the frequency range of 0.1 to 10 Hz (Fig. 2 a, b). Furthermore, our evaluation of the storage (G’) and loss (G’’) moduli of the two hydrogels revealed a limited correlation at strains ranging from 0.1–10% (Figure S7a, b), indicating their resistance to external shear forces. Additionally, the comparison of G’ and G’’ values between Gel Nap-TGY and Gel Nap-TGY + SB demonstrated subtle variations in mechanical and elastic properties, suggesting alterations in the behavior of Nap-TGY following the assembly of SB. Furthermore, Gel Nap-AGY was synthesized by SPSS, and rheological analysis indicated that the storage modulus (G’) was significantly higher than the loss modulus (G’’) within the investigated strain (0.01-1%) and frequency (0.1–10 Hz) ranges (Figure S8a, b). These findings support the characterization of the samples as hydrogels with resilient networks. In order to further elucidate the rheological properties of Nap-TGY and Nap-TGY + SB gels, circular dichroism (CD) was employed to analyze their protein secondary structures. As illustrated in Figure S7c,d, the circular dichroism spectra of Gel Nap-TGY and Gel Nap-TGY + SB displayed a positive peak at 220 nm and a negative peak at 232 nm, indicative of the β-sheet secondary structure in both hydrogels [ 24 ]. Furthermore, the CD spectrum of Gel Nap-TGY exhibited a positive peak at a wavelength of 245 nm, likely attributed to the tyrosine residue, with a similar variation observed in Gel Nap-TGY + SB. Transmission electron microscopy (TEM) was utilized to analyze the intricate structures of both hydrogels. The TEM images displayed the dense nanofiber networks present in the Gel Nap-TGY, with a diameter measuring 55.5 ± 4.5 nm, as depicted in Fig. 2 c, d. Following incorporation of SB, the TEM images of the Gel Nap-TGY + SB exhibited an increased number of small black granules within the nanofibers, with average diameters of 63.6 ± 10.4 nm. These characterization findings indicated that SB effectively co-assembled with Nap-TGY within the supramolecular hydrogel, as opposed to mere physical mixing. Cumulative release of SB from the Gel Nap-TGY + SB in vitro Although Nap-TGY hydrogels have demonstrated the capacity to encapsulate or load drugs, their potential to enhance drug retention or extend the duration of drug action remains unclear. To address this issue, our initial investigation focused on the in vitro release of SB from Nap-TGY + SB hydrogels. The release kinetics of SB were analyzed using HPLC, and the cumulative release profile was divided into two distinct stages: rapid release (stage 1, days 1–5) and sustained slow release (stage 2, days 5–10). During the rapid release phase, drug release assays indicated that the cumulative amount of SB released was 33.4 ± 3.8% on day 5, which was significantly higher than the 7.5 ± 1.7% release observed on day 1 in vitro (Fig. 3 a). The release rate of SB exhibited an initial sharp increase within the first 5 days, followed by a gradual decline during phase 2. By day 10, the average cumulative release of SB from the Gel Nap-TGY + SB reached 39.7 ± 4.7%. The findings from stages 1 and 2 indicate that the Gel Nap-TGY + SB not only protected the drug from rapid release but also extended its slow and sustained release in vitro. These results support the potential suitability of the Gel Nap-TGY + SB for in vivo investigations of anti-inflammatory therapy following MI/R. In addition, to assess the potential selective disassembly of Gel Nap-TGY + SB by phosphorylated p38, the hydrogels were exposed to LPS-induced macrophage culture supernatant (SN). The extracted mixed supernatants were also subjected to HPLC analysis at different incubation times (as mentioned above). Figure 3 a clearly demonstrated a sustained release of SB202190 from Gel Nap-TGY + SB. Over the course of 5 days, the cumulative amount of released SB202190 increased rapidly from 19.3 ± 2.2% on day 1 to 50.3 ± 2.7%. Evidently, the total quantity of released SB was significantly higher than that released by Gel Nap-TGY + SB alone. These results strongly suggest that Gel Nap-TGY + SB can be selectively disassembled by phosphorylated p38, leading to the release of SB in a controllable manner. Evaluation of Gel Nap-TGY and Gel Nap-AGY cytotoxicity To assess the impact of Gel Nap-TGY and the control Nap-AGY on cell viability, RAW264.7 macrophages and H9C2 cells were treated with varying concentrations of the hydrogels for 24 and 48 h. The CCK8 assay indicated that the addition of Nap-TGY to the culture media at concentrations ranging from 6.25 to 200 µM did not result in a significant inhibition of RAW264.7 macrophage viability (Figure S9a), as well as H9c2 cells (Figure S9c). It is worth noting that at the same concentration, Nap-AGY demonstrated no significant cytotoxic effects on the viability of both cell types after 24 and 48 hours of intervention (Figure S9b and S9d). These findings provide evidence that both Nap-TGY and Nap-AGY hydrogelators are non-cytotoxic and suitable for use in both in vivo and in vitro studies. p38-instructed phosphorylation of Gel Nap-TGY Following the characterization of Gel Nap-TGY and Gel Nap-TGY + SB, an assessment of their p38-activated gel-to-sol transition properties was conducted. Specifically, macrophages were treated with LPS (1 µg/ml) for 24 h to extract lysates containing phosphorylated p38 [ 25 ]. The lysate and hydrogel were incubated overnight for subsequent detection. HPLC and HR-MS analyses were used to elucidate the chemical changes in the hydrogels as they transitioned from gel to solution following p38 phosphorylation. The HPLC results presented in Figure S10 clearly indicate that the peak value of Gel Nap-TGY exhibited a forward shift after co-incubation with cell lysate. According to result of HR-MS (Figure S11), Nap-TGY yielded Nap-T(p)GY(p). Specifically, the transition of Gel Nap-TGY to the hydrophilic product Nap-T(p)GY(p) in the presence of phosphorylated p38 results in a structural transformation from microscopic nanofibers to nanoparticles within the hydrogel, facilitating the release of SB. Gel Nap-TGY + SB regulated the phosphorylation of p38 MAPK in LPS-induced macrophages P38 MAPK is widely expressed throughout the body, with six identified subtypes, including p38α1/α2, p38β1/β2, p38γ, and p38δ. Among these subtypes, p38α and p38β are highly expressed, particularly in the heart and brain, and are known to be key protein kinases in inflammation, cell differentiation, and apoptosis [ 14 , 26 ]. However, research on hydrogels targeting p38 for anti-inflammatory therapies remains limited. In this study, RAW264.7 macrophages were utilized to assess the levels of p38 MAPK phosphorylation induced by LPS stimulation. Initially, varying doses (25 µM, 50 µM, 100 µM) of Gel Nap-TGY or Gel Nap-AGY were added into the cell culture medium for a duration of 2 h, with a corresponding concentration of 10 µM of SB [ 27 ]. Subsequently, the medium was replaced with LPS (1 µg/ml) for co-culture, resulting in an increase in p38 phosphorylation (Fig. 4 a). Following treatment with varying concentrations of Gel Nap-TGY and Gel Nap-AGY, the levels of total and phosphorylated p38 protein were assessed using western blotting. The results depicted in Figure S12a and S12b demonstrate a notable decrease in p38 phosphorylation after a 2-h pretreatment with Gel Nap-TGY, suggesting its potential inhibitory effect on p38 phosphorylation. Furthermore, the lowest expression of phosphorylated p38 was observed at a Nap-TGY concentration of 100 µM. In contrast, pretreatment with Gel Nap-AGY did not significantly inhibit p38 phosphorylation. Additionally, no statistically significant difference was observed between the Gel Nap-AGY pretreatment group and the LPS group (Figure S12). These results suggest that despite the structural similarity between Nap-TGY and Nap-AGY, which differ by only one amino acid residue, their effects on p38 phosphorylation vary significantly. This discrepancy prompted the hypothesis that the varying outcomes may be due to the divergent mechanisms of action of the two hydrogels upon cellular internalization. It has been proposed that the TGY amino acid sequence, comprising Threonine, Glycine, and Tyrosine, may represent a specific target for the inhibition of p38 phosphorylation [ 28 , 29 ]. To elucidate the effect of Gel Nap-TGY on p-p38, various concentrations were used to assess protein expression (Fig. 4 c, e, f). Western blot analysis revealed a significant reduction in p-p38 expression levels in macrophages treated with Gel Nap-TGY compared to those in the LPS group, with the most pronounced decrease observed at a concentration of 100 µM. Upon entry into cells, Nap-TGY appeared to suppress p38 activity, consequently reducing p-p38 expression. Our hypothesis suggests that following LPS-induced inflammation in macrophages, the accumulated intracellular phosphate ions interact with the hydrogel, leading to dual phosphorylation of the TGY motif and ultimately diminishing intracellular p-p38 production. This discovery emphasizes the significance of understanding the complex molecular pathways involved in the control of p38 activity, as well as the potential therapeutic efficacy of Gel Nap-TGY in modulating inflammatory reactions. The immune response is triggered by external or internal stimuli, and macrophages serve as key mediators of inflammation by upregulating inflammatory factors and governing defense and immune modulation processes [ 30 ]. Thus, we examined the impact of Gel Nap-TGY (100 µM), SB (10 µM), or Gel Nap-TGY + SB (100 µM) on the expression of p-p38 in co-cultured macrophages (Fig. 4 b, d). Two hours after co-culturing, the medium was replaced and LPS was added for a 24-h incubation period. Immunofluorescence assays revealed a significant increase in p-p38 levels in macrophages following LPS treatment, accompanied by noticeable changes in the expression of F4/80 (a macrophage marker), indicating marked phosphorylation of p38 in macrophages. In Nap-TGY hydrogel-treated macrophages that were co-cultured with LPS, there was a significant decrease in the ratio of p-p38 to F4/80 compared with that in the LPS group (Fig. 4 d). Correspondingly, SB was shown to be involved in the dephosphorylation of p38, although its efficacy was not as pronounced as that of Gel Nap-TGY + SB. Cell experiments demonstrated that the prolonged release of SB from Gel Nap-TGY + SB markedly enhanced its ability to dephosphorylate p38 in LPS-stimulated macrophages. Our study indicated that the presence of a single hydrogel led to a decrease in p-p38 levels, whilst loading of the gel with SB further enhanced this effect, implying its potential dephosphorylation and anti-inflammatory capabilities. Gel Nap-TGY injection and retention in ischemic myocardium Injectable hydrogels are commonly utilized as delivery systems in biological investigations pertaining to cardiac repair, serving as carriers for pharmaceutical agents [ 20 , 31 ]. To determine the retention and slow-release capabilities of Gel Nap-TGY + SB, we conducted an in vivo assessment of its localization in the ischemic myocardium. 5(6)-Carboxy-tetramethylrhodamine N-succinimidyl ester (TMR-NHS) was incorporated into Gel Nap-TGY. A total of 100 µL of TMR-NHS were administered to the ischemic myocardium of MI/R rats, which formed the TMR group. Hydrogel-assembled TMR was administered to other rats in the same manner to form the Gel-TMR group. At 1 h, 1 d, 3 d, and 5 d post-surgery, the major organs, including the heart, were excised, and the fluorescence intensity was assessed using a small animal imager. Our analysis revealed that the fluorescence intensity of the hearts in both experimental groups was notably high within the first hour and on the first day after surgery (Fig. 3 b ) . Interestingly, the fluorescence signal persisted in the Gel-TMR group on postoperative days 3 and 5, suggesting that the dye incorporated into Gel Nap-TGY remained detectable for a minimum of 5 days, whereas the fluorescence signal in the TMR group markedly decreased over time. In the Gel-TMR group, strong fluorescence was detected in the heart and liver up to 3 d post-surgery, signifying hepatic metabolism of the dye, whereas fluorescence in the kidneys was not evident until the fifth day (Figure S13). Conversely, the liver and kidneys, organs responsible for excretion of the TMR group exhibited strong signals, indicating that rhodamine dye without a hydrogel coating was readily metabolized. These findings suggest that Gel Nap-TGY has distinct adhesive characteristics that may prolong the retention of loaded substances in vivo, along with a sustained release effect, suggesting its potential to extend the therapeutic efficacy of drugs. Injection of the Gel Nap-TGY + SB regulated p38 MAPK phosphorylation and the inflammatory response Recovery of damaged cardiomyocytes and implementation of anti-inflammatory therapy are crucial for restoration of infarcted myocardium. Based on the promising outcomes mentioned above, our subsequent experiments focused on determining whether Gel Nap-TGY could augment the suppressive impact of SB on p-p38 elevation triggered by MI/R injury in an animal model. Given the propensity of MI to significantly promote inflammation, an MI/R rat model was induced by temporarily occluding the left coronary artery for 45 min, followed by reperfusion, in order to evaluate the effectiveness of Gel Nap-TGY + SB in vivo. To evaluate the impact of the hydrogel on MI/R, a series of experiments were conducted, as outlined in Fig. 5 a. Inhibition of the P38-related signaling pathway effectively ameliorates MI/R injury [ 8 , 32 ]. To investigate the temporal pattern of increased p-p38, which is indicative of peak inflammation following MI/R, rat ischemic myocardial tissues were collected at various time points (days 1, 3, 7, 14, 21, 28) and were analyzed for p-p38 expression using western blotting (Figure S14a-c). The results demonstrated a gradual increase in p-p38 expression from day 1 post-MI/R, a peak on day 7, followed by a subsequent decrease in levels. These results were in line with those of previous research that the expression of p-p38 increases during the initial phase of inflammation and decreases as inflammation subsided, indicating a strong correlation with the onset of inflammation [ 33 ]. Consequently, day 7 was chosen as the time point for assessing the impact of the hydrogel therapy. Effective management of the inflammatory response after MI/R is essential for facilitating tissue repair [ 34 ]. Therefore, this study examined the effect of Gel Nap-TGY + SB on p-p38-mediated inflammation. Following the administration of different hydrogels or SB into the ischemic myocardium of MI/R rats on day 7, a notable reduction in p-p38 expression levels was observed in the Gel Nap-TGY + SB group compared with the MI/R group (Fig. 5 b-f). However, no significant statistical differences were observed between the Gel Nap-TGY and the SB groups. Additionally, we examined the impact of Gel Nap-TGY or Gel Nap-TGY + SB on the expression of p-Erk1/2, which has a similar molecular structure to p38 MAPK. Western blotting (Fig. 5 e, f) revealed that the expression level of p-Erk1/2 was unaffected by either hydrogel at 7 d post-myocardial injection. This phenomenon can be explained by the substitution of the TEY amino acid residues for TGY in the molecular structure of Erk1/2, as TEY is incapable of acting as a competing substrate for Nap-TGY and preventing phosphorylation. Furthermore, sera from each group of rats were collected and analyzed using ELISA kits on days 1, 3, and 7 post-MI/R. In response to stress, p38 MAPK phosphorylates and regulates the activity of various downstream targets, such as the NF-κB transcription factor, resulting in the transcription and release of inflammatory factors, including tumor necrosis factor-α (TNF-α) [ 35 ]. TNF-α serves as a robust pro-inflammatory cytokine and has the ability to recruit immune cells, modulate vascular permeability, and facilitate tissue injury. As shown in Fig. 5 g-j, on the first and third day after MI/R, the levels of inflammatory cytokines IL-1β and TNF-α were significantly increased compared with those in the sham group, while the expression of the pro-inflammatory factor MCP-1 peaked on day 7. Additionally, the levels of G-CSF, a cytokine that inhibits myocardial apoptosis and tissue repair, were notably decreased in rats subjected to MI/R surgery [ 36 ]. The ELISA results demonstrated that the injection of Gel Nap-TGY or Gel Nap-TGY + SB into the ischemic myocardium of rats resulted in significantly lower expression levels of inflammatory cytokines compared with those in the MI/R group. Additionally, MCP-1 levels decreased, whereas G-CSF levels increased, suggesting that these treatments effectively suppressed the escalation of inflammatory and exerted anti-inflammatory effects in the context of MI/R. Interestingly, the expression levels of IL-1β, TNF-α and G-CSF did not show significant differences compared to those in the MI/R group at all time points in the SB group. These results suggest that a single injection of SB may not effectively inhibit the increase in inflammatory cytokines following MI/R injury. This effect was particularly evident in the significantly reduced levels of p38 phosphorylation (Fig. 5 b-d). Moreover, Gel Nap-TGY has been shown to improve inflammation following myocardial ischemia, with its anti-inflammatory properties further augmented through the co-assembly of SB within the hydrogel and subsequent sustained release. Gel Nap-TGY + SB reduced phosphorylation of p38 MAPK in ischemic myocardium, and hydrogel treatment may reduce apoptosis To investigate the potential impacts of Gel Nap-TGY + SB on the ischemic myocardium, we conducted immunofluorescence analysis. Given the hypothesis that the mitigation of inflammation could be attributed to the diminished p38 MAPK phosphorylation, we assessed the quantity of phosphorylated-p38-positive (p-p38 + ) cells relative to cardiac troponin T (cTnT) in the border zone of infarcted hearts day 7 post-MI/R injury. Following the administration of various hydrogels or pharmaceutical agents to the injured myocardium, co-immunofluorescence analysis of p-p38 (green) and cTnT (red) was conducted to assess the effect of Gel Nap-TGY + SB on suppressing p38 phosphorylation. Examination of heart slices revealed a higher number of p-p38 + cells in the MI/R group compared with that in the sham group (Fig. 6 a). Quantitative analysis of p-p38 and cTnT ratios using immunofluorescence indicated that there was no significant difference in the population of p-p38 positive cells between the SB injection and MI/R group (Fig. 6 c). Additionally, administration of Gel Nap-TGY and Gel Nap-TGY + SB led to a partial decrease in the quantity of p-p38 positive cells, with the most substantial decline observed in the Gel Nap-TGY + SB group. This suggests that the TGY amino acid moiety in Gel Nap-TGY may undergo degelatinization in response to the generation of phosphate ions by p38, consequently preventing the phosphorylation of p38. The continuous apoptosis of cardiomyocyte following ischemic-reperfusion injury can reduce the cardiomyocyte population and facilitate the development of myocardial fibrosis [ 8 , 34 ]. Hypoxia and inflammation accelerate apoptosis during the initial phases of injury. Specifically, cleaved caspase3, which is the active form of caspase3, plays a crucial role in programmed cell death and apoptosis. Suppressing of cleaved caspase3 has been shown to protect cardiomyocytes from apoptosis induced by ischemia or hypoxia [ 37 ]. Bax, a pro-apoptotic protein, serves as an antagonist of the anti-apoptotic protein Bcl-2, and these proteins exert opposite functions [ 38 ]. A reduction in the Bcl-2/Bax ratio leads to increased mitochondrial membrane permeability and apoptosis [ 39 ]. The levels of apoptotic molecules (Bax, Bcl-2, caspase3, and cleaved caspase3) were analyzed using western blotting on day 3 post-MI/R (Fig. 6 b, d-g). As anticipated, a notable reduction in cleaved caspase3 and significant increase in the Bcl-2/Bax ratio were observed in Gel Nap-TGY + SB group, suggesting a potential anti-apoptotic effect. However, these results were not observed in the Gel Nap-TGY and SB groups. These results are consistent with those obtained through ELISA, suggesting that the Gel Nap-TGY + SB not only suppressed the inflammatory response triggered by p38 phosphorylation but also alleviated cell apoptosis in the ischemic myocardium of MI/R rats. Gel Nap-TGY + SB improved cardiac function of MI/R rats Next, we investigated the hypothesis that the release of SB from the Nap-TGY + SB dual-inhibition hydrogel could provide myocardial protection for ischemic heart repair. Echocardiography was chosen to evaluate cardiac function due to its non-invasive nature. The efficacy of the treatment for cardiac repair was assessed using echocardiography on day 28 post-MI/R (Fig. 7 a). Compared with that in the sham group, we observed a significant decrease in left ventricular ejection fraction (LVEF) and fractional shortening (FS) in the MI/R group, indicating cardiac dysfunction. However, rats in the MI/R group treated with hydrogel or SB solution exhibited notable improvements in cardiac function, particularly in the Gel Nap-TGY + SB group. Echocardiographic assessments revealed that the LVEF and FS values in the Gel Nap-TGY + SB group increased from 41.0 ± 2.8% to 73.2 ± 2.9% and from 21.2 ± 1.6% to 43.5 ± 2.8%, respectively, compared with those in the MI/R group (Fig. 7 b, c). The left ventricular internal diameter at end diastole (LVIDd) and left ventricular internal diameter at end systole (LVIDs) in the Gel Nap-TGY + SB group decreased from 10.2 ± 2.3 mm to 6.9 ± 1.2 mm and 8.0 ± 1.8 mm to 4.2 ± 0.8 mm, respectively, compared with those in MI/R rats (Fig. 7 d, e). Conversely, although the LVEF and FS values of the Gel Nap-TGY and SB solution groups were significantly higher than those of the MI/R group, their cardioprotective effects were notably inferior to those observed after Gel Nap-TGY + SB treatment. Notably, treatment with SB solution resulted in only a slight increase in LVEF and FS, along with a slight decrease in the thickness of LVIDd and LVIDs (Fig. 7 b-e). These findings suggest that Gel Nap-TGY + SB exerts better cardioprotective properties and therapeutic effects compared with those of either Gel Nap-TGY or SB alone following MI/R injury. Gel Nap-TGY + SB attenuated ventricular remodeling, increased angiogenesis in rat ischemic myocardium post MI/R In addition to inflammation, myocardial fibrosis is a significant factor in ventricular remodeling following MI, resulting in thinning of the ventricular wall, increased heart volume, and chronic heart failure [ 40 , 41 ]. Myocardial fibrosis and ventricular wall thickness are key indicators used to assess post-MI remodeling. Masson's trichrome staining on day 28 post-MI/R revealed myocardial fibrosis in all rats (Figure S15). As shown in Fig. 8 a, histological analysis revealed blue-stained regions indicative of fibrosis in the ischemic ventricle of MI/R rats, accompanied by a reduction in left ventricular thickness compared with that in the sham group. Consistent with echocardiographic assessments, treatment with Gel Nap-TGY + SB reduced fibrotic area size and ventricular wall thinning. Specifically, Nap-TGY + SB treatment resulted in a reduction in scar size from 40.8 ± 4.1% to 20.6 ± 6.3% and an increase in ventricular wall thickness from 900.8 ± 116.6 µm to 2125 ± 271.0 µm compared with those in the MI/R group (Fig. 8 c, d). The Gel Nap-TGY and SB treatments resulted in a decrease in the area of myocardial fibrosis, but with lower efficacy than the Gel Nap-TGY + SB treatment. We also hypothesized that a reduction in myocardial fibrosis promotes angiogenesis, ultimately resulting in cardiac function recovery. To test this hypothesis, we conducted immunostaining of vascular endothelium in ischemic rat hearts on day 28 post-MI/R using CD31 (an endothelial cell marker) and vWF (a marker for microvessels) (Fig. 8 b). As shown in Fig. 8 e, the percentage of CD31-positive cells per field was 2.0 ± 0.4 in the sham group and 2.3 ± 0.5 in the MI/R group, and it increased to 5.9 ± 0.9 in the Gel Nap-TGY + SB group. Moreover, the expression levels of CD31 in the Gel Nap-TGY and SB groups were measured at 4.5 ± 0.6 and 2.5 ± 0.8, respectively. Correspondingly, the vWF positive areas (%) in the MI/R group was 3.0 ± 1.0, whereas in the Gel Nap-TGY and SB groups were 4.5 ± 0.5 and 3.1 ± 0.6, respectively. Interestingly, the vWF positive areas in the combined Gel Nap-TGY + SB treatment group increased to 5.2 ± 1.3. Furthermore, the results demonstrated that the proangiogenic effects of Gel Nap-TGY + SB were significantly greater than those of the individual treatments with Gel Nap-TGY or SB solution. This indicates that Gel Nap-TGY + SB may have a substantial impact on promoting angiogenesis in ischemic myocardial tissue. Assessment of organ toxicity and biocompatibility of Gel Nap-TGY + SB Given that the hydrogelator compounds Nap-TGY and SB are released into the systemic circulation, ensuring biosafety is crucial for clinical translation. In response to address this, a thorough examination of potential pathological changes in multiple organs is needed. However, the safety of hydrogel for use as an effective treatment for MI/R rats remains uncertain. To assess the biological safety of Gel Nap-TGY + SB, we used hematoxylin-eosin (HE) reagent to stain the major organs of rats 28 days post MI/R operation. Our findings indicated no significant pathological alterations in the main viscera of treated rats (Fig. 9 a), providing direct evidence of the safety of Gel Nap-TGY + SB. In addition, comprehensive biochemical assays were performed to assess the systemic effects of Gel Nap-TGY + SB. At day 28 after last treatment, serum alanine aminotransferase (ALT) (Fig. 9 b), aspartate aminotransferase (AST) (Fig. 9 c), and blood urea nitrogen (BUN) (Fig. 9 d) were measured and found to be within the normal range in the Sham group and the four experimental groups. In summary, histologic and biochemical analyses demonstrated that Gel Nap-TGY + SB has good biocompatibility and non-toxicity in vivo. These results strongly confirm the potential value of Gel Nap-TGY as a safe and highly efficacious drug system for MI/R therapy. Conclusion This study illustrated that Gel Nap-TGY possesses the capability to inhibit the phosphorylation of p38, exhibits favorable biocompatibility, and is injectable. This hydrogel can effectively encapsulate small-molecule drugs and enable their controlled release. SB was successfully incorporated into the Nap-TGY hydrogel to form Gel Nap-TGY + SB, leveraging its ability to inhibit p38 activity. Based on these findings, the efficacy of SB and Nap-TGY in facilitating cardiac repair in ischemic hearts was substantiated by administration of a specially formulated Gel Nap-TGY + SB to a rat MI/R model. In addition, in vitro experiments confirmed the ability of the Gel Nap-TGY + SB to inhibit the phosphorylation of p38. Finally, injection of the Gel Nap-TGY + SB suppressed p38 activity in the ischemic region, ultimately resulting in anti-inflammatory, anti-apoptotic, and pro-angiogenic effects (Fig. 1 ). This study is first to demonstrate the therapeutic potential of the Gel Nap-TGY + SB for cardiac repair through the inhibition of p38 MAPK activation in the ischemic myocardium. Our findings suggest that a reduction in inflammation can be achieved through dual inhibition of p38, a mechanism that may have implications for various forms of cardiac protection. Furthermore, this dual-function hydrogel offers a unique perspective for clinical management and treatment strategies in other diseases. Materials and methods Synthesis, and preparation of the Gel Nap-TGY + SB The hydrogelator Nap-TGY was synthesized using solid phase peptide synthesis. High-resolution mass spectrometry (HR-MS), 1 H nuclear magnetic resonance (NMR), and 13 C nuclear magnetic resonance (NMR) were used to characterize the compound after its purification via high-performance liquid chromatography (HPLC). As a next step, the gelling capacity of Nap-TGY was tested using the heating-cooling protocol. One thousand microliters of PBS containing 10 mg Nap-TGY were prepared. The mixture was then heated to 65°C and sonicated until it became clear after it reached a pH of 8.0. Within 30 minutes of cooling the solution to room temperature (25°C), a clarified hydrogel with a concentration of 1.0wt% was formed. Similarly, a transparent Gel Nap-TGY + SB was prepared by heating up/cooled down SB202190 (Selleck Biotechnology Co., Ltd, United States). In the procedure described above, control hydrogelator Nap-AGY and diphosphorylated hydrogelator Nap-T(p)GY(p) were synthesized trans. Animals myocardial I/R model The 8–10 weeks, weighing 200-220g Male Sprague Dawley rats were purchased from Anhui medical University experiment animal center. All animals were given free food and water. The experimental approach complies with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals (Ethical Approval Number: LLSC20221128). Myocardial ischemia-reperfusion (MI/R) model rat was used in this experiment. Each rat was anesthetized with pentobarbital sodium (40 mg/kg), its trachea was intubated and connected to a ventilator (Small Animal Ventilator, RWD Life science, Shenzhen, China), which was secured to the operating table. The skin of rats was separated layer by layer, the heart was exposed by thoracotomy, and the left anterior descending branch was ligated with 6 − 0 non-resorbable nylon suture. After 45 minutes of ligation, the sutures would be disengaged and continuously perfused until response observation time point. Rat hydrogel treatment The rats were randomly divided into five groups: Sham, MI/R, Gel Nap-TGY, SB, and MI/R + Gel Nap-TGY + SB. After 40 min of sustained ischemia in rat coronary arteries, a total volume of 100 µl Gel Nap-TGY (1.0wt%), mixed with SB (10 µM), was injected at 5 points along the border zones of ischemic myocardial (20 µl at each point) sites for the Gel Nap-TGY + SB group. The equal amount of Nap-TGY hydrogel (1.0wt%) was injected into the rat heart in Gel Nap-TGY group. Rats receiving 100 µl SB202190 (10 µM) were set up as SB group. As with the Sham group, they were only given the same surgical procedure, and the left ventricular muscle was injected with 100 µl of saline instead of being ligated. In addition, the MI/R rats were injected with 100 µl of saline to serve as controls. In each group, the ligatures were released after five minutes of cardiac stability following injection, ensuring 45 minutes of ischemia. Surgical incisions were stitched after complete ischemia and hemostasis, respectively. During reperfusion, the animals were allowed to awaken naturally on a warm insulation blanket and receive food and water freely. Enzyme linked immunosorbent assay For the purpose of evaluating and testing Gel Nap-TGY + SB on anti-inflammation, inflammatory cytokines were employed as markers at the cellular level. Post-operatively, MI/R rats were bled intravenously on days 1, 3, and 7 after the above intervention. In compliance with manufacturer's instructions, the rat enzyme-linked immunosorbent assay (ELISA) Kits for interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein (MCP)-1 were measured as directed. The ELISA kits for IL-1β (Cat. No: EK301B/4–96), TNF-α (Cat. No: EK382/3–96), and MCP-1 (Cat. No: EK387-96) were purchased from Multi- sciences Co., Ltd, and G-CSF (Cat. No: ZY-E61349R) was purchased from Sanghai Zeye Biotech Co., Ltd. Animals immunoblotting Ischemia myocardial tissue was extracted and lysed with Radio Immunoprecipitation Assay buffer (RIPA), protease and phosphatase inhibitor cocktail (50X) and 1mM PMSF (Beyotime, Beijing, China). Cracking of the cardiac tissue was performed with an automatic sample rapid grinding apparatus (Cat. No. JXFSTPRP-24L, Shanghai Jingxin Co., Ltd, China). The protein-containing lysates obtained in the previous procedure were centrifuged at 12,000 g for 15 min at 4 ℃, and the concentration of protein supernatant was measured by BCA protein quantification (Solarbio, Beijing, China), and 30 µg tissue lysates were electrophoresed in the 10% SDS page gel (Shanghai Epizyme Biomedical Technology Co., Ltd). Based on the previous method [ 42 ], western blotting was performed. The primary antibodies used for the incubation process include total p38 MAPK, Phospho-p38 MAPK, total Erk MAPK, Phospho-Erk MAPK, total/cleaved caspase3, GAPDH, as mentioned in Supporting Information. Immunofluorescence analysis At day 7 postoperatively, the five groups of rats were anesthetized and hearts were removed. The expression level of phosphorylated p38, a factor associated with inflammation, was measured according to the immunofluorescence after MI/R. The hearts of the rats after treating with or without hydrogels were first fixed with 4% paraformaldehyde for 24 h, dehydrated by different gradients (20%, 30%, 40%) of sucrose for 72 h, and then embedded by optimal cutting temperature compound (OCT; SAKURA, USA) and placed in -80 ℃ refrigerator for further processing. The embedded heart tissue was placed in the pathological frozen microtome (FS800, RWD Life science Co. Ltd., China) for continuous coronal frozen sectioning with a thickness of 10 µm. The sections were then fixed in 4% paraformaldehyde for 30 min, permeabilized in 0.3% triton X-100 (Solarbio Life Science Co. Ltd., China) diluted in PBS for 20 min, and blocked in 5% BSA in PBS for 30 min at room temperature, which is identical to the basic immunofluorescence manipulation. Afterwards, the sections were incubated in the primary antibody reagent overnight at 4°C, followed by incubation in Alexa Fluor 568 immunoglobulin G (IgG) [heavy and light chains (H&L)] (1:1000) and Alexa Fluor 488 IgG (H&L) (1:1000) for 2 h at room temperature. Primary antibodies used in this study included anti-phospho-p38 MAPK from Cell Signaling Technology (9212s) and anti-cardiac troponin T/cTnT from Proteintech Group (15513-1-AP). After three rinses with PBS, the tissue staining was further stained with DAPI (2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride) and visualized using a full slide scanning system. The fluorescence intensity of p-p38 (green) in the cardiomyocyte (cTnT staining, red) was counted using ImageJ software and the ratio of p-p38 to cTnT was calculated. In each group, five samples were tested. Echocardiography As reported previously [ 43 ], the cardiac function of rats was examined by echocardiography on day 28 following MI/R following injection with saline, Gel Nap-TGY, SB and Gel Nap-TGY + SB. The rats were anesthetized with inhalation of isoflurane (1.5 to 2%) in O 2 , and their chest hair was removed with a small animal shaving device. With a 23-MHz transducer, an M-mode ultrasound was performed using a Vinno 6 imaging system (VINNO Technology Co. Ltd., Suzhou, China). M-mode tracings were used to detect left ventricular ejection fraction (LVEF) and fractional shortening (FS). Short-axis and long-axis views were used to determine the left ventricular internal dimension diastole (LVIDd) and left ventricular internal dimension systole (LVIDs). The experiment was conducted double-blind, with the sonographer unaware of the experimental group and the results analyzed by a third party. Infarct detection Having been measured by echocardiography, the rats were anesthetized, and the hearts were removed and washed. After perfusing the hearts with PBS, the hearts were fixed for 24 h at 4°C with 4% paraformaldehyde. Hearts were then embedded in paraffin and pathological sections were performed with a paraffin microtome (HM340E Thermo Fisher Scientific, MA, USA). The sections were cut into 4 µm at midpapillary muscle level for Masson's trichrome. The area of infarction and degree of fibrosis were measured with Masson's trichrome staining. The microscopic images were analyzed at 40 × amplification, and the scar area and LV thickness were measured with ImageJ. Histological analysis The CD31 and vWF immunohistochemistry staining was performed to investigate the effect of Gel Nap-TGY + SB on the levels of angiogenic factors and angiogenesis in vivo. The total number of CD31 + cells and VEGF + area at the site of ischemic myocardium were counted in 10 to 15 randomly selected fields from five different heart slides in each group. The protocol of immunohistochemistry staining for heart cryosections was the same as previously reported [ 43 ]. After the treatment of Gel Nap-TGY + SB, SB, Gel Nap-TGY at day 28 after injection, liver, spleen, lung, and kidney slices were stained with Hematoxylin and Eosin Staining solution (Beyotime Tech, Shanghai, China) to determine the overall morphology of tissues and analyze the histotoxicity. Statistical analysis Data are presented as means ± SEM from at least three independent experiments. Statistical significance between two groups was determined by Student’s t-test. Statistical differences among three or more groups were compared by using one-way analysis of variance (ANOVA), followed by Bonferroni post hoc test. P value of less than 0.05 was considered statistically significant (* P < 0.05, ** P < 0.01, *** P < 0.001). All statistical analyses were performed with GraphPad Prism software (version 8.0.1). Declarations Supporting information: Scheme S1-S2 and Figure S1-S15. Acknowledgments Funding: This work was supported by the National Natural Science Foundation of China (Grant No. 82470281, No. 81970231), the National Natural Science Foundation of Anhui Province (No. 2308085MH238, 2208085MH198), and Postgraduate Innovation Research and Practice Program of Anhui Medical University (No. YJS20230134). Author Contributions Di Wang, Aoxue Xu, and Haitao Su contributed equally to this work. Yaguang Wang, and Ye Zhang designed the study. D.W., A.X., and H.S. performed the cells and animal experiments. Y-P.Z. and Y.W. designed the hydrogels and conducted assembly experiment. D.W. wrote original manuscript. A.X., H.S., L.J., G.L. revised the manuscript. Y.W., G.L, and Y.Z. were responsible for the conception, design and supervision of the study.All authors contributed to the manuscript preparation and revision. Data availability No datasets were generated or analysed during the current study. Ethics approval and consent to participate All experimental protocols and procedures were reviewed and approved by Anhui Medical University's Ethics Committee (LLSC20221128), and followed the Guide for the Care and Use of Laboratory Animals. Consent for publication All authors of this study agreed to publish. Competing interests The authors declare that they have no competing interests. References Yellon DM, Hausenloy DJ: Myocardial reperfusion injury. N Engl J Med 2007, 357: 1121-1135. 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Wang Y, Wang D, Wu C, Wang B, He S, Wang H, Liang G, Zhang Y: MMP 9-instructed assembly of bFGF nanofibers in ischemic myocardium to promote heart repair. Theranostics 2022, 12: 7237-7249. Wang D, Hu Y, Zhang L, Cai H, Wang Y, Zhang Y: Dual delivery of an NF-κB inhibitor and IL-10 through supramolecular hydrogels polarizes macrophages and promotes cardiac repair after myocardial infarction. Acta Biomater 2023, 164: 111-123. Additional Declarations No competing interests reported. Supplementary Files SustainedReleaseofDualInhibitionSI.docx Supporting Information Supporting information: Scheme S1-S2 and Figure S1-S15. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5944861","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":412091126,"identity":"c1f058f2-35f3-4014-aa32-95ad003fd714","order_by":0,"name":"Di Wang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Di","middleName":"","lastName":"Wang","suffix":""},{"id":412091127,"identity":"16ec39ef-f9ea-4634-b733-aea259e6fb58","order_by":1,"name":"Aoxue Xu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Aoxue","middleName":"","lastName":"Xu","suffix":""},{"id":412091129,"identity":"d704d94d-ccc2-41b9-806b-8bf7482ea498","order_by":2,"name":"Haitao Su","email":"","orcid":"","institution":"The Second Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Haitao","middleName":"","lastName":"Su","suffix":""},{"id":412091131,"identity":"a647c5fc-9227-4e53-99a8-84e130dcc7b9","order_by":3,"name":"Youpei Zhang","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Youpei","middleName":"","lastName":"Zhang","suffix":""},{"id":412091132,"identity":"36c1fb05-ed6a-48a9-8d76-9b7b311ab3cd","order_by":4,"name":"Lingling Jiang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lingling","middleName":"","lastName":"Jiang","suffix":""},{"id":412091134,"identity":"79f453c4-6731-4cda-92f5-c18db7cdbfdf","order_by":5,"name":"Yaguang Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYJADxgeMDWCGAdFamA1I1sImQZQWg+NnD7/mqalN7J/dfq3i545tiQ3szdskGGru4NZyJi/NmufY8cQZd86U3ew9czuxgedYmQTDsWc4tZgdyDEz5mE7lthwIyftBm8bUItEjhnQhYdxazn/Bqjl37HE+UAthX9BWuTfENByI8f4MW9bTeKGG+nHmCG28ODXYn/jjRnj3L4Dxhtv5DBLy7bdNm7jSSu2SDiGW4tkf47xhzff6mTn3Uh/+PFt223ZfvbDG298qMGtBQjYpHgYQAp4INHBBiIS8GkARvrHHwx1QJr9AX51o2AUjIJRMGIBAClBYXwcD6cXAAAAAElFTkSuQmCC","orcid":"","institution":"The Second Affiliated Hospital of Anhui Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yaguang","middleName":"","lastName":"Wang","suffix":""},{"id":412091144,"identity":"2b8c6521-75ab-4839-b048-ba15f89c8128","order_by":6,"name":"Gaolin Liang","email":"","orcid":"","institution":"Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Gaolin","middleName":"","lastName":"Liang","suffix":""},{"id":412091145,"identity":"e70ef6ae-2271-48da-86e7-57667312ce88","order_by":7,"name":"Ye Zhang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-02-02 08:23:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5944861/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5944861/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75740571,"identity":"ac50ad22-7f0b-4772-b2b7-0321db71198b","added_by":"auto","created_at":"2025-02-07 16:26:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":267871,"visible":true,"origin":"","legend":"\u003cp\u003eThe schematic illustration of how Gel Nap-TGY+SB promotes cardiac protection after MI/R. a) Chemical structure of Gel Nap-TGY+SB. b) After the hydrogel was injected into the myocardial infarction site of MI/R model rats, it slowly released SB202190, reduced inflammation, and enhanced myocardium repair.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5944861/v1/b63e9c519e05b839e58896fc.png"},{"id":75740570,"identity":"b647877e-2f3b-435f-9856-4e53cfe8a936","added_by":"auto","created_at":"2025-02-07 16:26:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":81053,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of the hydrogels. Storage (G’) and loss (G’’) moduli of the Nap-TGY (a) and Nap-TGY+SB hydrogels (b) at different frequencies, with a strain of 1.0%. Transmission electron microscopy (TEM) images of the nanofibers in the Nap-TGY (c) and Nap-TGY+SB hydrogels (d). Insets are photographs of the Nap-TGY and Nap-TGY+SB hydrogels.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5944861/v1/5265404005fff9844e61f339.png"},{"id":75740705,"identity":"f5217877-fbdd-4e99-ba05-c861221fa03e","added_by":"auto","created_at":"2025-02-07 16:34:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":64884,"visible":true,"origin":"","legend":"\u003cp\u003eRelease effect of Gel Nap-TGY+SB in vitro and retention time in vivo. a) Cumulative release curve of SB in culture medium after incubation of Gel Nap-TGY+SB in PBS (0.01 M, pH 7.4) and the cell supernatant (from LPS-induced macrophages) at different time points, respectively. b) Fluorescence imaging of rats after heart resection at 1 h, 1 d, 3 d, and 5 d post-MI/R. Data are presented as mean ± SEM, n = 3. Statistical significance was assessed using Tukey’s post-test with one-way ANOVA. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5944861/v1/fd607c5560c039f6760aff1d.png"},{"id":75740582,"identity":"cae69ec5-ed73-4540-a9f5-3b8bf2546f34","added_by":"auto","created_at":"2025-02-07 16:26:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":106662,"visible":true,"origin":"","legend":"\u003cp\u003ePhosphorylation of p38 MAPK was altered following the administration of Gel Nap-TGY+SB in LPS-stimulated macrophages. a) Schematic illustration of cell experiments. b) Immunofluorescence images showing the expression of p-p38+ of F4/80+. Scale bar = 20 µm. c) WB analysis of p-p38 and p38 levels in the total cellular protein for the six groups (n = 3). d) Quantification of the fluorescence intensity of p-p38+ of F4/80+ by ImageJ software. e, f) Quantitative analysis of p-p38 and p38 expression levels using ImageJ software. Data are shown as means ± SEM of three independent experiments. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns, no statistical difference.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5944861/v1/959d1b31a1b23e9013681016.png"},{"id":75740707,"identity":"abd7a5a0-b390-4328-880a-044e0e76a9c8","added_by":"auto","created_at":"2025-02-07 16:34:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":85757,"visible":true,"origin":"","legend":"\u003cp\u003eGel Nap-TGY+SB reduced inflammation in vivo. a) Schematic illustration of the establishment of the MI/R rat model and the therapeutic process. b) Western blotting analysis of p-p38 and p-Erk1/2 levels in the ischemic myocardial tissue protein for the five groups (n = 4). c-f) Quantification of p-p38 and p-Erk1/2 in b). g-j) Quantitative analysis results of the expression levels of inflammatory factors (IL-1β, TNF-α, G-CSF and MCP-1) in the different treatment groups (n = 5 for each group) at day 1, 3, 7 post MI/R. Data are shown as means ± SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns, no statistical significance.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5944861/v1/da3baeae7289d676b1071d3d.png"},{"id":75740575,"identity":"43847066-d904-4ef7-b020-8db28a686b79","added_by":"auto","created_at":"2025-02-07 16:26:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":154917,"visible":true,"origin":"","legend":"\u003cp\u003eSB202190/NapFFTGY hydrogel regulated inflammation and apoptosis in vivo. a) Representative immunofluorescence staining images for p-p38\u003csup\u003e+\u003c/sup\u003e of cTnT\u003csup\u003e+\u003c/sup\u003e in the border region of the ischemic myocardium of rats 7 days post-MI (n = 5). Scale bars: 50 µm. b) Western blotting analysis of Bax, Bcl-2, cleaved caspase3, and caspase3 levels in the ischemic myocardial tissues from the five groups (n = 4). c) Quantitative analysis of p-p38 in the positive ratio of cTnT in a). d-g) Quantitative analysis of the Bcl2-/Bax ratio and cleaved caspase3 and caspase3 expression levels using ImageJ software. Data are shown as means ± SEM. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns, no statistical significance.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5944861/v1/1b5e6a441bdfcc9af906a636.png"},{"id":75740572,"identity":"92511ed5-02b5-462a-9021-3566d9935cb6","added_by":"auto","created_at":"2025-02-07 16:26:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":66559,"visible":true,"origin":"","legend":"\u003cp\u003eNon-invasive echocardiography of heart function in rats 28 days post-MI/R following the different treatments.\u003cstrong\u003e \u003c/strong\u003ea) Representative mode echocardiograms for each group (n = 6). b-e) Quantitative analyses of the LVEF, FS, LVIDd, and LVIDs for each group. Data are presented as the mean ± SEM for the EF (%), FS (%), LVIDd (mm), and LVIDs (mm). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns was considered as not statistically significant.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5944861/v1/c726a124a79efe5f29bc3ff1.png"},{"id":75740595,"identity":"08d22533-6eb0-48a3-a356-28120aa0c010","added_by":"auto","created_at":"2025-02-07 16:26:16","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":160126,"visible":true,"origin":"","legend":"\u003cp\u003eGel Nap-TGY+SB attenuated fibrotic remodeling of the infarcted ventricle and increased vascularization in the ischemic area 28 days post-MI/R. a) Representative images of Masson's trichrome staining heart sections of rats after MI/R (n = 6). Scale bars: 1 mm for heart cross-sections and 100 µm for enlarged images of the infarcted area. b) Immunochemistry staining of CD31 and vWF in heart sections from rats in each group after MI/R. (n = 6). Scale bar: 50 µm. c-d) Quantification analyses of fibrosis area and left ventricular (LV) wall thickness of the MI/R zone in each group. e) Quantification of the percentage of CD31 staining. f) Quantification of the percentage of vWF staining. Data are presented as the mean ± SEM of the CD31 positive areas (%), vWF positive areas (%), fibrotic area (%), and left ventricular wall thickness (mm). *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns, no statistical significance.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5944861/v1/90cb5003cc771d0fa31446e2.png"},{"id":75740590,"identity":"f136c18f-1090-41b0-a498-a9fa4f67bace","added_by":"auto","created_at":"2025-02-07 16:26:16","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":212807,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of organ toxicity and biocompatibility of Gel Nap-TGY+SB. (a) H\u0026amp;E staining of major organs from rats sacrificed at day 28 post-MI/R. Different treatment including sham, saline, Gel Nap-TGY, SB, Gel Nap-TGY+SB intramyocardial injection. Scar bar: 100 µm. b–d The concentrations of ALT (b), AST (c), and BUN (d) in each serum from rat treated with Gel Nap-TGY+SB and no treatment. Data are expressed as mean ± SEM.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5944861/v1/e47abfca12ed25e296d990ec.png"},{"id":78251605,"identity":"91e924d9-161f-4dcd-83f7-4b61108f5b14","added_by":"auto","created_at":"2025-03-11 10:02:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4142973,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5944861/v1/a0941662-ca26-45e5-a862-1d1739d40cbe.pdf"},{"id":75740706,"identity":"799bde8f-59c1-488e-a622-05523f2d87ec","added_by":"auto","created_at":"2025-02-07 16:34:15","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4292573,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupporting Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupporting information: Scheme S1-S2 and Figure S1-S15.\u003c/p\u003e","description":"","filename":"SustainedReleaseofDualInhibitionSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-5944861/v1/61e76df55cbdc3a107feabac.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sustained Release of Dual p38 Inhibitors via Supramolecular Hydrogels to Enhance Cardiac Repair after MI/R Injury","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTimely implementation of interventions aimed at revascularization can effectively reduce perioperative mortality in patients with myocardial infarction (MI). However, myocardial reperfusion may also result in myocardial ischemia-reperfusion (MI/R) injury, leading to cardiac cell death [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. MI/R injury encompasses cardiac hypertrophy, myocardial fibrosis, deformation of the heart chamber, as well as reduced diastolic and systolic functions, ultimately resulting in the development of heart failure [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFollowing MI/R, a sequence of inflammatory cascades ensures within the ischemic myocardium, ultimately resulting in further injury. This process involves inflammatory cells infiltration, particularly of neutrophils and monocytes, and subsequent secretion of chemokines and cytokines [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The activation of toll-like receptors (TLR)-mediated pathways, the complement system, and reactive oxidative species (ROS) generation by pro-inflammatory signals, along with the modulation of mitogen-activated protein kinase (MAPK)-related pathways, expedites the inflammatory response. Inhibiting the TLR4/MyD88/NF-κB pathway or reducing ROS production can mitigate inflammation and myocardial fibrosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Early treatment to reduce inflammation can help prevent negative changes in the heart structure.\u003c/p\u003e \u003cp\u003eP38 MAPKs are a family of stress-activated kinases, together with extracellular signal-regulated kinases 1/2 (Erk1/2), c-Jun N-terminal Kinases (JNKs), extracellular signal-regulated kinases (Erk5), which constitute the four branches of the MAPK system [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The activation of this system has been implicated in various pathological stress responses within the body. Moreover, activation of p38 MAPK is linked to various heart conditions, such as dilated cardiomyopathy, heart attacks, and fibrotic cardiomyopathy [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Recent studies on p38 MAPK have primarily focused on pharmacological approaches and revealed that inhibiting p38 may enhance myocardial contractility and mitigate MI/R injury [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTraditionally, p38 is believed to be dual phosphorylated by upstream dual-specific MAPK kinases (MAPKKs) such as MAP Kinase Kinase 3 (MKK3) and MAP Kinase Kinase 6 (MKK6) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. P38 MAPK is known to exist in four isoforms (α, β, γ, δ), all of which contain the Thr-Gly-Tyr (TGY) amino acid sequence. Activation of p38 requires dual phosphorylation at Thr180 and Tyr182 sites [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Crystal structure analyses have revealed that the specificity of p38 MAPK is due to the Gly residue in the Thr-Gly-Tyr (TGY) motif, distinguishing it from other MAPK pathway molecules, such as Erk and JNK [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSB202190, a compound that inhibits the p38 MAPK pathway by binding to the ATP-binding site of p38 MAPK and reducing its activity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Previous studies have shown that SB202190 improves heart function, decreases heart inflammation, and prevents heart cell death in both organisms and laboratory setting [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Its specificity makes it a valuable tool for studying cell signaling pathways linked to p38 overexpression, especially in the context of inflammation therapy. SB202190 exhibits strong hydrophobic properties and requires frequent administration in animal studies. Therefore, choosing an appropriate carrier for targeted delivery to the inflammatory site is important for improving its effectiveness.\u003c/p\u003e \u003cp\u003eRecent studies have demonstrated that hydrogel can enhance the solubility and absorption rate of insoluble drugs, improve targeting, decrease adverse reactions, and enable more precise release compared with traditional drug carriers [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. To address these inherent challenges, the present study investigates the utilization of a biocompatible hydrogel loaded with SB202190 (SB) for controlled release in the infarcted region, with the aim of modulating inflammation and reducing ischemia-reperfusion injury. Given the structural similarity between the hydrogel and the phosphorylation binding site and the known inhibitory effects of SB on p38 MAPK phosphorylation, this approach holds promise as a potential dual-action therapy for myocardial ischemia.\u003c/p\u003e \u003cp\u003eIn this study, we first developed a p38 Mark kinase-triggered gel-to-solution transition strategy for local delivery and controlled release of p38 inhibitors in the ischemic myocardium to ameliorate the pro-inflammatory microenvironment. Specifically, we rationally designed a hydrogelator, Nap-Phe-Phe-Thr-Gly-Tyr-OH (Nap-TGY), consisting of i) a commonly used self-assembling motif Nap-Phe-Phe, and ii) a dipeptide, Thr-Gly-Tyr-OH (TGY), for p38-recognized phosphorylation. Supramolecular hydrogel Nap-TGY (Gel Nap-TGY) was easily obtained via a heating-cooling method. The p38 inhibitor SB was co-assembled with Nap-TGY to yield the hydrogel Nap-TGY\u0026thinsp;+\u0026thinsp;SB (Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB). We propose that, upon p38 activation, Nap-TGY in the hydrogels is efficiently converted to its hydrophilic phosphate, Nap-Phe-Phe-Thr (H2PO3)-Gly-Tyr(H2PO3) (Nap-TpGYp), triggering the disassembly of the hydrogels and the release of SB in a sustainable manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). After the hydrogel is locally injected, p38 responsive release of SB at the ischemic myocardium site is achieved (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In turn, the released SB downregulates p38 activity and consequently inhibits its downstream proteins to efficiently improve the inflammatory microenvironment.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHydrogels preparation and characterization\u003c/h2\u003e \u003cp\u003eInitially, a solid-phase peptide (SPSS) was utilized to synthesize the hydrogelator Nap-TGY and the control hydrogelator Nap-Phe-Phe-Ala-Gly-Tyr-OH (Nap-AGY) (Scheme S1 and Scheme S2, Supporting Information). The molecular structures of Nap-TGY and Nap-AGY were confirmed by high-resolution mass spectrometry (HR-MS), \u003csup\u003e1\u003c/sup\u003eH nuclear magnetic resonance (\u003csup\u003e1\u003c/sup\u003eH NMR), and \u003csup\u003e13\u003c/sup\u003eC NMR spectroscopy (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S6).\u003c/p\u003e \u003cp\u003eSubsequently, the physical properties of Gel Nap-TGY, Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB, and Gel Nap-AGY were assessed through a series of experiments. The dynamic strain scanning characteristics of the three hydrogels were examined using a rheometer. At a strain amplitude of 1.0%, the storage modulus (G\u0026rsquo;) of Gel Nap-TGY and Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB were notably higher than their loss modulus (G\u0026rsquo;\u0026rsquo;) within the frequency range of 0.1 to 10 Hz (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). Furthermore, our evaluation of the storage (G\u0026rsquo;) and loss (G\u0026rsquo;\u0026rsquo;) moduli of the two hydrogels revealed a limited correlation at strains ranging from 0.1\u0026ndash;10% (Figure S7a, b), indicating their resistance to external shear forces. Additionally, the comparison of G\u0026rsquo; and G\u0026rsquo;\u0026rsquo; values between Gel Nap-TGY and Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB demonstrated subtle variations in mechanical and elastic properties, suggesting alterations in the behavior of Nap-TGY following the assembly of SB. Furthermore, Gel Nap-AGY was synthesized by SPSS, and rheological analysis indicated that the storage modulus (G\u0026rsquo;) was significantly higher than the loss modulus (G\u0026rsquo;\u0026rsquo;) within the investigated strain (0.01-1%) and frequency (0.1\u0026ndash;10 Hz) ranges (Figure S8a, b). These findings support the characterization of the samples as hydrogels with resilient networks. In order to further elucidate the rheological properties of Nap-TGY and Nap-TGY\u0026thinsp;+\u0026thinsp;SB gels, circular dichroism (CD) was employed to analyze their protein secondary structures. As illustrated in Figure S7c,d, the circular dichroism spectra of Gel Nap-TGY and Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB displayed a positive peak at 220 nm and a negative peak at 232 nm, indicative of the β-sheet secondary structure in both hydrogels [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Furthermore, the CD spectrum of Gel Nap-TGY exhibited a positive peak at a wavelength of 245 nm, likely attributed to the tyrosine residue, with a similar variation observed in Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB.\u003c/p\u003e \u003cp\u003eTransmission electron microscopy (TEM) was utilized to analyze the intricate structures of both hydrogels. The TEM images displayed the dense nanofiber networks present in the Gel Nap-TGY, with a diameter measuring 55.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5 nm, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d. Following incorporation of SB, the TEM images of the Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB exhibited an increased number of small black granules within the nanofibers, with average diameters of 63.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.4 nm. These characterization findings indicated that SB effectively co-assembled with Nap-TGY within the supramolecular hydrogel, as opposed to mere physical mixing.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCumulative release of SB from the Gel Nap-TGY + SB in vitro\u003c/h3\u003e\n\u003cp\u003eAlthough Nap-TGY hydrogels have demonstrated the capacity to encapsulate or load drugs, their potential to enhance drug retention or extend the duration of drug action remains unclear. To address this issue, our initial investigation focused on the in vitro release of SB from Nap-TGY\u0026thinsp;+\u0026thinsp;SB hydrogels. The release kinetics of SB were analyzed using HPLC, and the cumulative release profile was divided into two distinct stages: rapid release (stage 1, days 1\u0026ndash;5) and sustained slow release (stage 2, days 5\u0026ndash;10). During the rapid release phase, drug release assays indicated that the cumulative amount of SB released was 33.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8% on day 5, which was significantly higher than the 7.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7% release observed on day 1 in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The release rate of SB exhibited an initial sharp increase within the first 5 days, followed by a gradual decline during phase 2. By day 10, the average cumulative release of SB from the Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB reached 39.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7%. The findings from stages 1 and 2 indicate that the Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB not only protected the drug from rapid release but also extended its slow and sustained release in vitro. These results support the potential suitability of the Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB for in vivo investigations of anti-inflammatory therapy following MI/R.\u003c/p\u003e \u003cp\u003eIn addition, to assess the potential selective disassembly of Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB by phosphorylated p38, the hydrogels were exposed to LPS-induced macrophage culture supernatant (SN). The extracted mixed supernatants were also subjected to HPLC analysis at different incubation times (as mentioned above). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea clearly demonstrated a sustained release of SB202190 from Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB. Over the course of 5 days, the cumulative amount of released SB202190 increased rapidly from 19.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2% on day 1 to 50.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.7%. Evidently, the total quantity of released SB was significantly higher than that released by Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB alone. These results strongly suggest that Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB can be selectively disassembled by phosphorylated p38, leading to the release of SB in a controllable manner.\u003c/p\u003e\n\u003ch3\u003eEvaluation of Gel Nap-TGY and Gel Nap-AGY cytotoxicity\u003c/h3\u003e\n\u003cp\u003eTo assess the impact of Gel Nap-TGY and the control Nap-AGY on cell viability, RAW264.7 macrophages and H9C2 cells were treated with varying concentrations of the hydrogels for 24 and 48 h. The CCK8 assay indicated that the addition of Nap-TGY to the culture media at concentrations ranging from 6.25 to 200 \u0026micro;M did not result in a significant inhibition of RAW264.7 macrophage viability (Figure S9a), as well as H9c2 cells (Figure S9c). It is worth noting that at the same concentration, Nap-AGY demonstrated no significant cytotoxic effects on the viability of both cell types after 24 and 48 hours of intervention (Figure S9b and S9d). These findings provide evidence that both Nap-TGY and Nap-AGY hydrogelators are non-cytotoxic and suitable for use in both in vivo and in vitro studies.\u003c/p\u003e\n\u003ch3\u003ep38-instructed phosphorylation of Gel Nap-TGY\u003c/h3\u003e\n\u003cp\u003eFollowing the characterization of Gel Nap-TGY and Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB, an assessment of their p38-activated gel-to-sol transition properties was conducted. Specifically, macrophages were treated with LPS (1 \u0026micro;g/ml) for 24 h to extract lysates containing phosphorylated p38 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The lysate and hydrogel were incubated overnight for subsequent detection. HPLC and HR-MS analyses were used to elucidate the chemical changes in the hydrogels as they transitioned from gel to solution following p38 phosphorylation. The HPLC results presented in Figure S10 clearly indicate that the peak value of Gel Nap-TGY exhibited a forward shift after co-incubation with cell lysate. According to result of HR-MS (Figure S11), Nap-TGY yielded Nap-T(p)GY(p). Specifically, the transition of Gel Nap-TGY to the hydrophilic product Nap-T(p)GY(p) in the presence of phosphorylated p38 results in a structural transformation from microscopic nanofibers to nanoparticles within the hydrogel, facilitating the release of SB.\u003c/p\u003e\n\u003ch3\u003eGel Nap-TGY + SB regulated the phosphorylation of p38 MAPK in LPS-induced macrophages\u003c/h3\u003e\n\u003cp\u003eP38 MAPK is widely expressed throughout the body, with six identified subtypes, including p38α1/α2, p38β1/β2, p38γ, and p38δ. Among these subtypes, p38α and p38β are highly expressed, particularly in the heart and brain, and are known to be key protein kinases in inflammation, cell differentiation, and apoptosis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, research on hydrogels targeting p38 for anti-inflammatory therapies remains limited. In this study, RAW264.7 macrophages were utilized to assess the levels of p38 MAPK phosphorylation induced by LPS stimulation. Initially, varying doses (25 \u0026micro;M, 50 \u0026micro;M, 100 \u0026micro;M) of Gel Nap-TGY or Gel Nap-AGY were added into the cell culture medium for a duration of 2 h, with a corresponding concentration of 10 \u0026micro;M of SB [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Subsequently, the medium was replaced with LPS (1 \u0026micro;g/ml) for co-culture, resulting in an increase in p38 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Following treatment with varying concentrations of Gel Nap-TGY and Gel Nap-AGY, the levels of total and phosphorylated p38 protein were assessed using western blotting. The results depicted in Figure S12a and S12b demonstrate a notable decrease in p38 phosphorylation after a 2-h pretreatment with Gel Nap-TGY, suggesting its potential inhibitory effect on p38 phosphorylation. Furthermore, the lowest expression of phosphorylated p38 was observed at a Nap-TGY concentration of 100 \u0026micro;M. In contrast, pretreatment with Gel Nap-AGY did not significantly inhibit p38 phosphorylation. Additionally, no statistically significant difference was observed between the Gel Nap-AGY pretreatment group and the LPS group (Figure S12). These results suggest that despite the structural similarity between Nap-TGY and Nap-AGY, which differ by only one amino acid residue, their effects on p38 phosphorylation vary significantly. This discrepancy prompted the hypothesis that the varying outcomes may be due to the divergent mechanisms of action of the two hydrogels upon cellular internalization. It has been proposed that the TGY amino acid sequence, comprising Threonine, Glycine, and Tyrosine, may represent a specific target for the inhibition of p38 phosphorylation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. To elucidate the effect of Gel Nap-TGY on p-p38, various concentrations were used to assess protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, e, f). Western blot analysis revealed a significant reduction in p-p38 expression levels in macrophages treated with Gel Nap-TGY compared to those in the LPS group, with the most pronounced decrease observed at a concentration of 100 \u0026micro;M. Upon entry into cells, Nap-TGY appeared to suppress p38 activity, consequently reducing p-p38 expression. Our hypothesis suggests that following LPS-induced inflammation in macrophages, the accumulated intracellular phosphate ions interact with the hydrogel, leading to dual phosphorylation of the TGY motif and ultimately diminishing intracellular p-p38 production. This discovery emphasizes the significance of understanding the complex molecular pathways involved in the control of p38 activity, as well as the potential therapeutic efficacy of Gel Nap-TGY in modulating inflammatory reactions.\u003c/p\u003e \u003cp\u003eThe immune response is triggered by external or internal stimuli, and macrophages serve as key mediators of inflammation by upregulating inflammatory factors and governing defense and immune modulation processes [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Thus, we examined the impact of Gel Nap-TGY (100 \u0026micro;M), SB (10 \u0026micro;M), or Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB (100 \u0026micro;M) on the expression of p-p38 in co-cultured macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, d). Two hours after co-culturing, the medium was replaced and LPS was added for a 24-h incubation period. Immunofluorescence assays revealed a significant increase in p-p38 levels in macrophages following LPS treatment, accompanied by noticeable changes in the expression of F4/80 (a macrophage marker), indicating marked phosphorylation of p38 in macrophages. In Nap-TGY hydrogel-treated macrophages that were co-cultured with LPS, there was a significant decrease in the ratio of p-p38 to F4/80 compared with that in the LPS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Correspondingly, SB was shown to be involved in the dephosphorylation of p38, although its efficacy was not as pronounced as that of Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB. Cell experiments demonstrated that the prolonged release of SB from Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB markedly enhanced its ability to dephosphorylate p38 in LPS-stimulated macrophages. Our study indicated that the presence of a single hydrogel led to a decrease in p-p38 levels, whilst loading of the gel with SB further enhanced this effect, implying its potential dephosphorylation and anti-inflammatory capabilities.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGel Nap-TGY injection and retention in ischemic myocardium\u003c/h2\u003e \u003cp\u003eInjectable hydrogels are commonly utilized as delivery systems in biological investigations pertaining to cardiac repair, serving as carriers for pharmaceutical agents [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. To determine the retention and slow-release capabilities of Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB, we conducted an in vivo assessment of its localization in the ischemic myocardium. 5(6)-Carboxy-tetramethylrhodamine N-succinimidyl ester (TMR-NHS) was incorporated into Gel Nap-TGY. A total of 100 \u0026micro;L of TMR-NHS were administered to the ischemic myocardium of MI/R rats, which formed the TMR group. Hydrogel-assembled TMR was administered to other rats in the same manner to form the Gel-TMR group. At 1 h, 1 d, 3 d, and 5 d post-surgery, the major organs, including the heart, were excised, and the fluorescence intensity was assessed using a small animal imager. Our analysis revealed that the fluorescence intensity of the hearts in both experimental groups was notably high within the first hour and on the first day after surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Interestingly, the fluorescence signal persisted in the Gel-TMR group on postoperative days 3 and 5, suggesting that the dye incorporated into Gel Nap-TGY remained detectable for a minimum of 5 days, whereas the fluorescence signal in the TMR group markedly decreased over time. In the Gel-TMR group, strong fluorescence was detected in the heart and liver up to 3 d post-surgery, signifying hepatic metabolism of the dye, whereas fluorescence in the kidneys was not evident until the fifth day (Figure S13). Conversely, the liver and kidneys, organs responsible for excretion of the TMR group exhibited strong signals, indicating that rhodamine dye without a hydrogel coating was readily metabolized. These findings suggest that Gel Nap-TGY has distinct adhesive characteristics that may prolong the retention of loaded substances in vivo, along with a sustained release effect, suggesting its potential to extend the therapeutic efficacy of drugs.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInjection of the Gel Nap-TGY + SB regulated p38 MAPK phosphorylation and the inflammatory response\u003c/h3\u003e\n\u003cp\u003eRecovery of damaged cardiomyocytes and implementation of anti-inflammatory therapy are crucial for restoration of infarcted myocardium. Based on the promising outcomes mentioned above, our subsequent experiments focused on determining whether Gel Nap-TGY could augment the suppressive impact of SB on p-p38 elevation triggered by MI/R injury in an animal model. Given the propensity of MI to significantly promote inflammation, an MI/R rat model was induced by temporarily occluding the left coronary artery for 45 min, followed by reperfusion, in order to evaluate the effectiveness of Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB in vivo. To evaluate the impact of the hydrogel on MI/R, a series of experiments were conducted, as outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003ea.\u003c/p\u003e \u003cp\u003eInhibition of the P38-related signaling pathway effectively ameliorates MI/R injury [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. To investigate the temporal pattern of increased p-p38, which is indicative of peak inflammation following MI/R, rat ischemic myocardial tissues were collected at various time points (days 1, 3, 7, 14, 21, 28) and were analyzed for p-p38 expression using western blotting (Figure S14a-c). The results demonstrated a gradual increase in p-p38 expression from day 1 post-MI/R, a peak on day 7, followed by a subsequent decrease in levels. These results were in line with those of previous research that the expression of p-p38 increases during the initial phase of inflammation and decreases as inflammation subsided, indicating a strong correlation with the onset of inflammation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Consequently, day 7 was chosen as the time point for assessing the impact of the hydrogel therapy.\u003c/p\u003e \u003cp\u003eEffective management of the inflammatory response after MI/R is essential for facilitating tissue repair [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, this study examined the effect of Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB on p-p38-mediated inflammation. Following the administration of different hydrogels or SB into the ischemic myocardium of MI/R rats on day 7, a notable reduction in p-p38 expression levels was observed in the Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB group compared with the MI/R group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-f). However, no significant statistical differences were observed between the Gel Nap-TGY and the SB groups. Additionally, we examined the impact of Gel Nap-TGY or Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB on the expression of p-Erk1/2, which has a similar molecular structure to p38 MAPK. Western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, f) revealed that the expression level of p-Erk1/2 was unaffected by either hydrogel at 7 d post-myocardial injection. This phenomenon can be explained by the substitution of the TEY amino acid residues for TGY in the molecular structure of Erk1/2, as TEY is incapable of acting as a competing substrate for Nap-TGY and preventing phosphorylation.\u003c/p\u003e \u003cp\u003eFurthermore, sera from each group of rats were collected and analyzed using ELISA kits on days 1, 3, and 7 post-MI/R. In response to stress, p38 MAPK phosphorylates and regulates the activity of various downstream targets, such as the NF-κB transcription factor, resulting in the transcription and release of inflammatory factors, including tumor necrosis factor-α (TNF-α) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. TNF-α serves as a robust pro-inflammatory cytokine and has the ability to recruit immune cells, modulate vascular permeability, and facilitate tissue injury. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eg-j, on the first and third day after MI/R, the levels of inflammatory cytokines IL-1β and TNF-α were significantly increased compared with those in the sham group, while the expression of the pro-inflammatory factor MCP-1 peaked on day 7. Additionally, the levels of G-CSF, a cytokine that inhibits myocardial apoptosis and tissue repair, were notably decreased in rats subjected to MI/R surgery [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The ELISA results demonstrated that the injection of Gel Nap-TGY or Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB into the ischemic myocardium of rats resulted in significantly lower expression levels of inflammatory cytokines compared with those in the MI/R group. Additionally, MCP-1 levels decreased, whereas G-CSF levels increased, suggesting that these treatments effectively suppressed the escalation of inflammatory and exerted anti-inflammatory effects in the context of MI/R. Interestingly, the expression levels of IL-1β, TNF-α and G-CSF did not show significant differences compared to those in the MI/R group at all time points in the SB group. These results suggest that a single injection of SB may not effectively inhibit the increase in inflammatory cytokines following MI/R injury. This effect was particularly evident in the significantly reduced levels of p38 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-d). Moreover, Gel Nap-TGY has been shown to improve inflammation following myocardial ischemia, with its anti-inflammatory properties further augmented through the co-assembly of SB within the hydrogel and subsequent sustained release.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGel Nap-TGY\u0026thinsp;+\u0026thinsp;SB reduced phosphorylation of p38 MAPK in ischemic myocardium, and hydrogel treatment may reduce apoptosis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the potential impacts of Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB on the ischemic myocardium, we conducted immunofluorescence analysis. Given the hypothesis that the mitigation of inflammation could be attributed to the diminished p38 MAPK phosphorylation, we assessed the quantity of phosphorylated-p38-positive (p-p38\u003csup\u003e+\u003c/sup\u003e) cells relative to cardiac troponin T (cTnT) in the border zone of infarcted hearts day 7 post-MI/R injury. Following the administration of various hydrogels or pharmaceutical agents to the injured myocardium, co-immunofluorescence analysis of p-p38 (green) and cTnT (red) was conducted to assess the effect of Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB on suppressing p38 phosphorylation. Examination of heart slices revealed a higher number of p-p38\u003csup\u003e+\u003c/sup\u003e cells in the MI/R group compared with that in the sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Quantitative analysis of p-p38 and cTnT ratios using immunofluorescence indicated that there was no significant difference in the population of p-p38 positive cells between the SB injection and MI/R group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Additionally, administration of Gel Nap-TGY and Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB led to a partial decrease in the quantity of p-p38 positive cells, with the most substantial decline observed in the Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB group. This suggests that the TGY amino acid moiety in Gel Nap-TGY may undergo degelatinization in response to the generation of phosphate ions by p38, consequently preventing the phosphorylation of p38.\u003c/p\u003e \u003cp\u003eThe continuous apoptosis of cardiomyocyte following ischemic-reperfusion injury can reduce the cardiomyocyte population and facilitate the development of myocardial fibrosis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Hypoxia and inflammation accelerate apoptosis during the initial phases of injury. Specifically, cleaved caspase3, which is the active form of caspase3, plays a crucial role in programmed cell death and apoptosis. Suppressing of cleaved caspase3 has been shown to protect cardiomyocytes from apoptosis induced by ischemia or hypoxia [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Bax, a pro-apoptotic protein, serves as an antagonist of the anti-apoptotic protein Bcl-2, and these proteins exert opposite functions [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. A reduction in the Bcl-2/Bax ratio leads to increased mitochondrial membrane permeability and apoptosis [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The levels of apoptotic molecules (Bax, Bcl-2, caspase3, and cleaved caspase3) were analyzed using western blotting on day 3 post-MI/R (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, d-g). As anticipated, a notable reduction in cleaved caspase3 and significant increase in the Bcl-2/Bax ratio were observed in Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB group, suggesting a potential anti-apoptotic effect. However, these results were not observed in the Gel Nap-TGY and SB groups. These results are consistent with those obtained through ELISA, suggesting that the Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB not only suppressed the inflammatory response triggered by p38 phosphorylation but also alleviated cell apoptosis in the ischemic myocardium of MI/R rats.\u003c/p\u003e\n\u003ch3\u003eGel Nap-TGY + SB improved cardiac function of MI/R rats\u003c/h3\u003e\n\u003cp\u003eNext, we investigated the hypothesis that the release of SB from the Nap-TGY\u0026thinsp;+\u0026thinsp;SB dual-inhibition hydrogel could provide myocardial protection for ischemic heart repair. Echocardiography was chosen to evaluate cardiac function due to its non-invasive nature. The efficacy of the treatment for cardiac repair was assessed using echocardiography on day 28 post-MI/R (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Compared with that in the sham group, we observed a significant decrease in left ventricular ejection fraction (LVEF) and fractional shortening (FS) in the MI/R group, indicating cardiac dysfunction. However, rats in the MI/R group treated with hydrogel or SB solution exhibited notable improvements in cardiac function, particularly in the Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB group. Echocardiographic assessments revealed that the LVEF and FS values in the Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB group increased from 41.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8% to 73.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9% and from 21.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6% to 43.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8%, respectively, compared with those in the MI/R group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, c). The left ventricular internal diameter at end diastole (LVIDd) and left ventricular internal diameter at end systole (LVIDs) in the Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB group decreased from 10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3 mm to 6.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 mm and 8.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 mm to 4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 mm, respectively, compared with those in MI/R rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed, e). Conversely, although the LVEF and FS values of the Gel Nap-TGY and SB solution groups were significantly higher than those of the MI/R group, their cardioprotective effects were notably inferior to those observed after Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB treatment. Notably, treatment with SB solution resulted in only a slight increase in LVEF and FS, along with a slight decrease in the thickness of LVIDd and LVIDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb-e). These findings suggest that Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB exerts better cardioprotective properties and therapeutic effects compared with those of either Gel Nap-TGY or SB alone following MI/R injury.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGel Nap-TGY\u0026thinsp;+\u0026thinsp;SB attenuated ventricular remodeling, increased angiogenesis in rat ischemic myocardium post MI/R\u003c/h2\u003e \u003cp\u003eIn addition to inflammation, myocardial fibrosis is a significant factor in ventricular remodeling following MI, resulting in thinning of the ventricular wall, increased heart volume, and chronic heart failure [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Myocardial fibrosis and ventricular wall thickness are key indicators used to assess post-MI remodeling. Masson's trichrome staining on day 28 post-MI/R revealed myocardial fibrosis in all rats (Figure S15). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea, histological analysis revealed blue-stained regions indicative of fibrosis in the ischemic ventricle of MI/R rats, accompanied by a reduction in left ventricular thickness compared with that in the sham group. Consistent with echocardiographic assessments, treatment with Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB reduced fibrotic area size and ventricular wall thinning. Specifically, Nap-TGY\u0026thinsp;+\u0026thinsp;SB treatment resulted in a reduction in scar size from 40.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1% to 20.6\u0026thinsp;\u0026plusmn;\u0026thinsp;6.3% and an increase in ventricular wall thickness from 900.8\u0026thinsp;\u0026plusmn;\u0026thinsp;116.6 \u0026micro;m to 2125\u0026thinsp;\u0026plusmn;\u0026thinsp;271.0 \u0026micro;m compared with those in the MI/R group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec, d). The Gel Nap-TGY and SB treatments resulted in a decrease in the area of myocardial fibrosis, but with lower efficacy than the Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB treatment.\u003c/p\u003e \u003cp\u003eWe also hypothesized that a reduction in myocardial fibrosis promotes angiogenesis, ultimately resulting in cardiac function recovery. To test this hypothesis, we conducted immunostaining of vascular endothelium in ischemic rat hearts on day 28 post-MI/R using CD31 (an endothelial cell marker) and vWF (a marker for microvessels) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee, the percentage of CD31-positive cells per field was 2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 in the sham group and 2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 in the MI/R group, and it increased to 5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 in the Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB group. Moreover, the expression levels of CD31 in the Gel Nap-TGY and SB groups were measured at 4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 and 2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8, respectively. Correspondingly, the vWF positive areas (%) in the MI/R group was 3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0, whereas in the Gel Nap-TGY and SB groups were 4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 and 3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6, respectively. Interestingly, the vWF positive areas in the combined Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB treatment group increased to 5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3. Furthermore, the results demonstrated that the proangiogenic effects of Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB were significantly greater than those of the individual treatments with Gel Nap-TGY or SB solution. This indicates that Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB may have a substantial impact on promoting angiogenesis in ischemic myocardial tissue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of organ toxicity and biocompatibility of Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB\u003c/h2\u003e \u003cp\u003eGiven that the hydrogelator compounds Nap-TGY and SB are released into the systemic circulation, ensuring biosafety is crucial for clinical translation. In response to address this, a thorough examination of potential pathological changes in multiple organs is needed. However, the safety of hydrogel for use as an effective treatment for MI/R rats remains uncertain. To assess the biological safety of Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB, we used hematoxylin-eosin (HE) reagent to stain the major organs of rats 28 days post MI/R operation. Our findings indicated no significant pathological alterations in the main viscera of treated rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea), providing direct evidence of the safety of Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB. In addition, comprehensive biochemical assays were performed to assess the systemic effects of Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB. At day 28 after last treatment, serum alanine aminotransferase (ALT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb), aspartate aminotransferase (AST) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec), and blood urea nitrogen (BUN) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed) were measured and found to be within the normal range in the Sham group and the four experimental groups. In summary, histologic and biochemical analyses demonstrated that Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB has good biocompatibility and non-toxicity in vivo. These results strongly confirm the potential value of Gel Nap-TGY as a safe and highly efficacious drug system for MI/R therapy.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study illustrated that Gel Nap-TGY possesses the capability to inhibit the phosphorylation of p38, exhibits favorable biocompatibility, and is injectable. This hydrogel can effectively encapsulate small-molecule drugs and enable their controlled release. SB was successfully incorporated into the Nap-TGY hydrogel to form Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB, leveraging its ability to inhibit p38 activity. Based on these findings, the efficacy of SB and Nap-TGY in facilitating cardiac repair in ischemic hearts was substantiated by administration of a specially formulated Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB to a rat MI/R model. In addition, in vitro experiments confirmed the ability of the Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB to inhibit the phosphorylation of p38. Finally, injection of the Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB suppressed p38 activity in the ischemic region, ultimately resulting in anti-inflammatory, anti-apoptotic, and pro-angiogenic effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This study is first to demonstrate the therapeutic potential of the Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB for cardiac repair through the inhibition of p38 MAPK activation in the ischemic myocardium. Our findings suggest that a reduction in inflammation can be achieved through dual inhibition of p38, a mechanism that may have implications for various forms of cardiac protection. Furthermore, this dual-function hydrogel offers a unique perspective for clinical management and treatment strategies in other diseases.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis, and preparation of the Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB\u003c/h2\u003e \u003cp\u003eThe hydrogelator Nap-TGY was synthesized using solid phase peptide synthesis. High-resolution mass spectrometry (HR-MS), \u003csup\u003e1\u003c/sup\u003eH nuclear magnetic resonance (NMR), and \u003csup\u003e13\u003c/sup\u003eC nuclear magnetic resonance (NMR) were used to characterize the compound after its purification via high-performance liquid chromatography (HPLC). As a next step, the gelling capacity of Nap-TGY was tested using the heating-cooling protocol. One thousand microliters of PBS containing 10 mg Nap-TGY were prepared. The mixture was then heated to 65\u0026deg;C and sonicated until it became clear after it reached a pH of 8.0. Within 30 minutes of cooling the solution to room temperature (25\u0026deg;C), a clarified hydrogel with a concentration of 1.0wt% was formed. Similarly, a transparent Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB was prepared by heating up/cooled down SB202190 (Selleck Biotechnology Co., Ltd, United States). In the procedure described above, control hydrogelator Nap-AGY and diphosphorylated hydrogelator Nap-T(p)GY(p) were synthesized trans.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eAnimals myocardial I/R model\u003c/h2\u003e \u003cp\u003eThe 8\u0026ndash;10 weeks, weighing 200-220g Male Sprague Dawley rats were purchased from Anhui medical University experiment animal center. All animals were given free food and water. The experimental approach complies with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals (Ethical Approval Number: LLSC20221128).\u003c/p\u003e \u003cp\u003eMyocardial ischemia-reperfusion (MI/R) model rat was used in this experiment. Each rat was anesthetized with pentobarbital sodium (40 mg/kg), its trachea was intubated and connected to a ventilator (Small Animal Ventilator, RWD Life science, Shenzhen, China), which was secured to the operating table. The skin of rats was separated layer by layer, the heart was exposed by thoracotomy, and the left anterior descending branch was ligated with 6\u0026thinsp;\u0026minus;\u0026thinsp;0 non-resorbable nylon suture. After 45 minutes of ligation, the sutures would be disengaged and continuously perfused until response observation time point.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRat hydrogel treatment\u003c/h2\u003e \u003cp\u003eThe rats were randomly divided into five groups: Sham, MI/R, Gel Nap-TGY, SB, and MI/R\u0026thinsp;+\u0026thinsp;Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB. After 40 min of sustained ischemia in rat coronary arteries, a total volume of 100 \u0026micro;l Gel Nap-TGY (1.0wt%), mixed with SB (10 \u0026micro;M), was injected at 5 points along the border zones of ischemic myocardial (20 \u0026micro;l at each point) sites for the Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB group. The equal amount of Nap-TGY hydrogel (1.0wt%) was injected into the rat heart in Gel Nap-TGY group. Rats receiving 100 \u0026micro;l SB202190 (10 \u0026micro;M) were set up as SB group. As with the Sham group, they were only given the same surgical procedure, and the left ventricular muscle was injected with 100 \u0026micro;l of saline instead of being ligated. In addition, the MI/R rats were injected with 100 \u0026micro;l of saline to serve as controls. In each group, the ligatures were released after five minutes of cardiac stability following injection, ensuring 45 minutes of ischemia. Surgical incisions were stitched after complete ischemia and hemostasis, respectively. During reperfusion, the animals were allowed to awaken naturally on a warm insulation blanket and receive food and water freely.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme linked immunosorbent assay\u003c/h2\u003e \u003cp\u003eFor the purpose of evaluating and testing Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB on anti-inflammation, inflammatory cytokines were employed as markers at the cellular level. Post-operatively, MI/R rats were bled intravenously on days 1, 3, and 7 after the above intervention. In compliance with manufacturer's instructions, the rat enzyme-linked immunosorbent assay (ELISA) Kits for interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein (MCP)-1 were measured as directed. The ELISA kits for IL-1β (Cat. No: EK301B/4\u0026ndash;96), TNF-α (Cat. No: EK382/3\u0026ndash;96), and MCP-1 (Cat. No: EK387-96) were purchased from Multi- sciences Co., Ltd, and G-CSF (Cat. No: ZY-E61349R) was purchased from Sanghai Zeye Biotech Co., Ltd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAnimals immunoblotting\u003c/h2\u003e \u003cp\u003eIschemia myocardial tissue was extracted and lysed with Radio Immunoprecipitation Assay buffer (RIPA), protease and phosphatase inhibitor cocktail (50X) and 1mM PMSF (Beyotime, Beijing, China). Cracking of the cardiac tissue was performed with an automatic sample rapid grinding apparatus (Cat. No. JXFSTPRP-24L, Shanghai Jingxin Co., Ltd, China). The protein-containing lysates obtained in the previous procedure were centrifuged at 12,000 g for 15 min at 4 ℃, and the concentration of protein supernatant was measured by BCA protein quantification (Solarbio, Beijing, China), and 30 \u0026micro;g tissue lysates were electrophoresed in the 10% SDS page gel (Shanghai Epizyme Biomedical Technology Co., Ltd). Based on the previous method [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], western blotting was performed. The primary antibodies used for the incubation process include total p38 MAPK, Phospho-p38 MAPK, total Erk MAPK, Phospho-Erk MAPK, total/cleaved caspase3, GAPDH, as mentioned in Supporting Information.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence analysis\u003c/h2\u003e \u003cp\u003eAt day 7 postoperatively, the five groups of rats were anesthetized and hearts were removed. The expression level of phosphorylated p38, a factor associated with inflammation, was measured according to the immunofluorescence after MI/R. The hearts of the rats after treating with or without hydrogels were first fixed with 4% paraformaldehyde for 24 h, dehydrated by different gradients (20%, 30%, 40%) of sucrose for 72 h, and then embedded by optimal cutting temperature compound (OCT; SAKURA, USA) and placed in -80 ℃ refrigerator for further processing. The embedded heart tissue was placed in the pathological frozen microtome (FS800, RWD Life science Co. Ltd., China) for continuous coronal frozen sectioning with a thickness of 10 \u0026micro;m. The sections were then fixed in 4% paraformaldehyde for 30 min, permeabilized in 0.3% triton X-100 (Solarbio Life Science Co. Ltd., China) diluted in PBS for 20 min, and blocked in 5% BSA in PBS for 30 min at room temperature, which is identical to the basic immunofluorescence manipulation. Afterwards, the sections were incubated in the primary antibody reagent overnight at 4\u0026deg;C, followed by incubation in Alexa Fluor 568 immunoglobulin G (IgG) [heavy and light chains (H\u0026amp;L)] (1:1000) and Alexa Fluor 488 IgG (H\u0026amp;L) (1:1000) for 2 h at room temperature. Primary antibodies used in this study included anti-phospho-p38 MAPK from Cell Signaling Technology (9212s) and anti-cardiac troponin T/cTnT from Proteintech Group (15513-1-AP). After three rinses with PBS, the tissue staining was further stained with DAPI (2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride) and visualized using a full slide scanning system. The fluorescence intensity of p-p38 (green) in the cardiomyocyte (cTnT staining, red) was counted using ImageJ software and the ratio of p-p38 to cTnT was calculated. In each group, five samples were tested.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eEchocardiography\u003c/h2\u003e \u003cp\u003eAs reported previously [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], the cardiac function of rats was examined by echocardiography on day 28 following MI/R following injection with saline, Gel Nap-TGY, SB and Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB. The rats were anesthetized with inhalation of isoflurane (1.5 to 2%) in O\u003csub\u003e2\u003c/sub\u003e, and their chest hair was removed with a small animal shaving device. With a 23-MHz transducer, an M-mode ultrasound was performed using a Vinno 6 imaging system (VINNO Technology Co. Ltd., Suzhou, China). M-mode tracings were used to detect left ventricular ejection fraction (LVEF) and fractional shortening (FS). Short-axis and long-axis views were used to determine the left ventricular internal dimension diastole (LVIDd) and left ventricular internal dimension systole (LVIDs). The experiment was conducted double-blind, with the sonographer unaware of the experimental group and the results analyzed by a third party.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eInfarct detection\u003c/h2\u003e \u003cp\u003eHaving been measured by echocardiography, the rats were anesthetized, and the hearts were removed and washed. After perfusing the hearts with PBS, the hearts were fixed for 24 h at 4\u0026deg;C with 4% paraformaldehyde. Hearts were then embedded in paraffin and pathological sections were performed with a paraffin microtome (HM340E Thermo Fisher Scientific, MA, USA). The sections were cut into 4 \u0026micro;m at midpapillary muscle level for Masson's trichrome. The area of infarction and degree of fibrosis were measured with Masson's trichrome staining. The microscopic images were analyzed at 40 \u0026times; amplification, and the scar area and LV thickness were measured with ImageJ.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eHistological analysis\u003c/h2\u003e \u003cp\u003eThe CD31 and vWF immunohistochemistry staining was performed to investigate the effect of Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB on the levels of angiogenic factors and angiogenesis in vivo. The total number of CD31\u003csup\u003e+\u003c/sup\u003e cells and VEGF\u003csup\u003e+\u003c/sup\u003e area at the site of ischemic myocardium were counted in 10 to 15 randomly selected fields from five different heart slides in each group. The protocol of immunohistochemistry staining for heart cryosections was the same as previously reported [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. After the treatment of Gel Nap-TGY\u0026thinsp;+\u0026thinsp;SB, SB, Gel Nap-TGY at day 28 after injection, liver, spleen, lung, and kidney slices were stained with Hematoxylin and Eosin Staining solution (Beyotime Tech, Shanghai, China) to determine the overall morphology of tissues and analyze the histotoxicity.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM from at least three independent experiments. Statistical significance between two groups was determined by Student\u0026rsquo;s t-test. Statistical differences among three or more groups were compared by using one-way analysis of variance (ANOVA), followed by Bonferroni post hoc test. \u003cem\u003eP\u003c/em\u003e value of less than 0.05 was considered statistically significant (*\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). All statistical analyses were performed with GraphPad Prism software (version 8.0.1).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eSupporting information: Scheme S1-S2 and Figure S1-S15.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding: This work was supported by the National Natural Science Foundation of China (Grant No. 82470281, No. 81970231), the National Natural Science Foundation of Anhui Province (No. 2308085MH238, 2208085MH198), and Postgraduate Innovation Research and Practice Program of Anhui Medical University (No. YJS20230134).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDi Wang, Aoxue Xu, and Haitao Su contributed equally to this work. Yaguang Wang, and Ye Zhang designed the study. D.W., A.X., and H.S. performed the cells and animal experiments. Y-P.Z. and Y.W. designed the hydrogels and conducted assembly experiment. D.W. wrote original manuscript. A.X., H.S., L.J., G.L. revised the manuscript. Y.W., G.L, and Y.Z. were responsible for the conception, design and supervision of the study.All authors contributed to the manuscript preparation and revision.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental protocols and procedures were reviewed and approved by Anhui Medical University\u0026apos;s Ethics Committee (LLSC20221128), and followed the Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors of this study agreed to publish.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYellon DM, Hausenloy DJ: \u003cstrong\u003eMyocardial reperfusion injury.\u003c/strong\u003e \u003cem\u003eN Engl J Med \u003c/em\u003e2007, \u003cstrong\u003e357:\u003c/strong\u003e1121-1135.\u003c/li\u003e\n\u003cli\u003eEckle T, Bertazzo J, Khatua TN, Fatemi Tabatabaei SR, Moori Bakhtiari N, Walker LA, Martino TA: \u003cstrong\u003eCircadian Influences on Myocardial Ischemia-Reperfusion Injury and Heart Failure.\u003c/strong\u003e \u003cem\u003eCirc Res \u003c/em\u003e2024, \u003cstrong\u003e134:\u003c/strong\u003e675-694.\u003c/li\u003e\n\u003cli\u003eOng SB, Hern\u0026aacute;ndez-Res\u0026eacute;ndiz S, Crespo-Avilan GE, Mukhametshina RT, Kwek XY, Cabrera-Fuentes HA, Hausenloy DJ: \u003cstrong\u003eInflammation following acute myocardial infarction: Multiple players, dynamic roles, and novel therapeutic opportunities.\u003c/strong\u003e \u003cem\u003ePharmacol Ther 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\u003cstrong\u003e164:\u003c/strong\u003e111-123.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Myocardial ischemia-reperfusion, supramolecular hydrogels, p38 mitogen-activated protein kinase, phosphorylation, dual inhibition.","lastPublishedDoi":"10.21203/rs.3.rs-5944861/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5944861/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eActivation of p38 mitogen-activated protein kinase plays an important role in the progression of ventricular muscle inflammation after myocardial ischemia-reperfusion (MI/R). The inhibition of p38 activation in ischemic myocardium can reduce ventricular muscle remodeling post-MI. However, owing to the dynamic change of p38 in ischemic myocardium after MI, the clinical therapeutic effect of p38 inhibitors is insufficient. Herein, we describe the design of a hydrogelator Nap-Phe-Phe-Thr-Gly-Tyr-OH (Nap-TGY) to coassemble the p38 inhibitor SB202190 (SB), a p38 responsive supramolecular hydrogel (Gel Nap-TGY+SB) for local administration and p38 responsive release of SB to efficiently improve the inflammatory microenvironment. Under the overexpression of p38 in ischemic myocardium, Nap-TGY in the hydrogel is phosphorylated to yield hydrophilic Nap-Phe-Phe-Thr(H2PO3)-Gly-Tyr(H2PO3) (Nap-TpGYp), triggering the disassembly of the hydrogel and a responsive release of the inhibitor. Injection of hydrogel into the ischemic myocardium significantly reduces p38 phosphorylation, mitigates inflammation, and enhances angiogenesis. These findings suggest a novel therapeutic strategy for ischemic cardiomyopathy through modulation of the p38 mitogen-activated protein kinase (MAPK) pathway.\u003c/p\u003e","manuscriptTitle":"Sustained Release of Dual p38 Inhibitors via Supramolecular Hydrogels to Enhance Cardiac Repair after MI/R Injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-07 16:26:10","doi":"10.21203/rs.3.rs-5944861/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f06fda4c-2799-4f50-bbce-d8b3c23dffd0","owner":[],"postedDate":"February 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-11T09:53:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-07 16:26:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5944861","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5944861","identity":"rs-5944861","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}