Preparation Of Small Molecule Peptide-drug Hydrogel And Its Application In The Treatment Of The Myocardial Ischemia-reperfusion Injury

Background:The overproduction of reactive oxygen species and the outbreak of aseptic inflammation are the leading causes of myocardial ischemia-reperfusion injury.Because of the complex mechanism between inflammation and reactive oxygen species,a single strategy to treat myocardial ischemia-reperfusion injury by only antiinflammatory response or anti-ROS is ineffective.Toll-like receptor 4(TLR4),one of the TLR receptors,is an important node linking reactive oxygen species and inflammation.ROS recruit and activate immune cells by activating TLR4,which generates ROS through the expression of NAPDH oxidase,thus forming a positive feedback ROS-inflammatory cycle.Previous studies have found that rhein has a strong inhibitory effect on the TLR4/NF-κB pathway,but the poor water solubility of rhein limits its application.Bisphenylalanine(FF)-based self-assembly of peptides has been widely used in life sciences due to its good biocompatibility,low cost,and ease of design and preparation.The introduction of this system provides the possibility of solving the rhein’s limitation.At the same time,to enhance the antioxidant capacity of the peptide,we introduced epigallocatechin gallic acid(EGCG)and finally obtained the target compound EGCG@Rh-gel.It is expected to achieve sustained treatment of myocardial ischemia-reperfusion injury through dual inhibition of ROS and inflammationObjective:Rhein polypeptide material(Rh-DFDFG-ss-ERGD)with good biocompatibility and enzyme responsiveness was synthesized,and a co-assembled hydrogel system of EGCG and Rh-gel(EGCG@Rh-gel)was constructed.The functional therapeutic effects and underlying mechanisms of the EGCG@Rh-gel co-assembled hydrogels were validated through in vitro and in vivo experiments.Methods:1.Rh-DFDFG-ss-ERGD and Nap-DFDFG-ss-ERGD were synthesized by solid-phase synthesis,verified by high-resolution mass spectrometry,and purified by HPLC.Then we confirmed the gel-forming ability of EGCG and Rh-DFDFG-ss-ERGD under the action of enzyme cleavage.A transmission electron microscope(TEM)was used to observe the change of the microstructure of the hydrogel before and after the addition of EGCG.Furthermore,we employed circular dichroism(CD)and Fourier transforms infrared spectroscopy(FTIR)to explore the structural basis.CCK8 verified the biocompatibility of the peptides.2.To construct a model of cellular hypoxia and reoxygenation.The experimental groups are Control,H/R,Nap-gel,Rh-gel,and EGCG@Rh-gel.The protective effect of the hydrogels on cells was verified by dead and alive(PI/AM)staining and the scavenging ability of the hydrogels on reactive oxygen species was confirmed by DHE and DCFH-DA staining.TUNEL staining verified the effect of the hydrogels on cell apoptosis.Western blot was used to verify the inhibitory effect of the hydrogels on the TLR4/NF-κB pathway,and the changes in the expression levels of inflammatory factors TNF-α,IL-6,and IL-1β were observed by qPCR.3.Build the mouse myocardial ischemia-reperfusion model.Then carry out in vivo imaging of small animals to verify the retention of the hydrogels in mice by wrapping indocyanine green(ICG)on 1,3,and 7 days,respectively.Mice were reperfused for 3 days.Hearts were taken and sliced.DHE and DCFH-DA staining were performed to observe the status of reactive oxygen species,and TUNEL staining was used to mark the apoptosis of cardiomyocytes.On the 5th day of reperfusion,the mouse hearts were taken and sliced,and the distribution of M1 and M2 macrophages was observed by immunofluorescence staining.After 28 days of reperfusion,cardiac ultrasound and Masson staining were performed to detect the cardiac function and fibrosis of the mice.Conclusion:1.The Rh-DFDFG-ss-ERGD polypeptide was successfully synthesized and solved the poor water solubility of rhein.EGCG can co-assemble with Rh-DFDFG-ss-ERGD under the action of reduced glutathione through hydrogen bonding and π-π stacking to form a more stable hydrogel(EGCG@Rh-gel).2.In vitro experiments showed that EGCG@Rh-gel could alleviate cell necrosis and apoptosis caused by hypoxia and reoxygenation through reactive oxygen species scavenging and inhibition of TLR4/NF-κB pathway.3.In vivo experiments show that EGCG@Rh-gel can achieve longer retention.At the same time,the scavenging of reactive oxygen species and the inhibition of inflammation reduce the decline of cardiac function and the apoptosis of cardiomyocytes caused by myocardial ischemia-reperfusion.