Study On The Efficacy And Mechanism Of Phlorizin In The Treatment Of Tissue Fibrosis Based On Biomechanical Pharmacology Theor

The human body is composed of cells and interstitium.The distribution of the interstitium in the human body presents a multi-level spatial structure,from the large fascia,peritoneum,to the fine extracellular matrix.These multi-level spatial interstitial distributions form an interconnected network,which can transmit matter,information,energy,etc.Some diseases occur in the change of interstitial structure,which is collectively referred to as interstitial diseases.Tissue fibrosis is a typical interstitial disease,which refers to a disease in which the inherent fibroblasts or pericytes in the tissue and a small number of bone marrow-derived fibroblasts are activated into myofibroblasts after stimulation.These myofibroblasts secrete a large amount of extracellular matrix,affecting tissue structure and material exchange,resulting in impaired tissue function.Because the course of tissue fibrosis disease is long and the prognosis is not very well,there are still few clinically targeted treatment drugs.The research of treating tissue fibrosis has always been a hot spot and difficult point for researchers.In interstitial diseases,changes in interstitial tissues can directly affect the mechanical environment of the body,and drugs can also cure diseases by changing the mechanical environment of tissues.Biomechanopharmacology is a new interdisciplinary discipline that combines biomechanics and pharmacology.In addition to the combined effect of biomechanical and pharmacological factors,biomechanopharmacology also includes pharmacological interventions of biomechanical factors to induce signals and the influence of biomechanical factors on pharmacokinetics and pharmacodynamics.In the description of the function of drugs or prescriptions in Chinese medicine,there are many concepts related to biomechanopharmacology,such as "invigorate the circulation of blood","promoting the circulation of qi","removing stasis",etc.Combining the above disciplinary characteristics and disease characteristics,we focus on a traditional Chinese medicine named lychee seed,which has the effect of promoting the circulation of qi,dispersing stasis,dispelling cold and relieving pain.A main active monomeric component in lychee seed,phlorizin,was selected to explore the efficacy and mechanism of phlorizin in the treatment of tissue fibrosis diseases,new biomechanopharmacology evaluation indicators,as well as the possibility of using interstitial therapy injection with intervaginal space injection(ISI)to treat interstitial diseases represented by tissue fibrosis.Methods1.Randomly divide the mice into 6 groups:control group,liver fibrosis model group,silibinin treated group(positive control),low dose of phlorizin treated group,medium dose of phlorizin treated group,and high dose of phlorizin treated group.A mouse liver fibrosis model was induced by intraperitoneal injection of carbon tetrachloride(CCl4)dissolved in olive oil(1:9,v/v).Start treated administration at the fifth week.After 8 weeks,the mice were killed for sampling.The efficacy of phlorizin was evaluated by histological indicators(H&E staining,Sirius red staining),serological indicators(HA,LN,ALT,AST),and biomechanopharmacological indicators(liver denstiy,cytoskeleton).2.High-throughput sequencing of mRNA and LncRNA from different groups of liver tissue.The results were annotated with GO functions and analyzed by KEGG pathway enrichment.Analyze the interaction relationship between LncRNA and mRNA,and predict LncRNA target genes.Sequencing results were validated by molecular docking technology and experiments for detecting iron content in tissues.3.Preparation of phlorizin nanoparticles:phlorizin phospholipid complexes are prepared first.The complex nanoparticles are then wrapped with hyaluronic acid.To explore the physical quality of nanoparticular,the morphology,particle size,electrical potential,UV,IR DSC,XRD,and drug released rate were characterized.4.The mice were randomly divided into 5 groups:control group,pulmonary fibrosis model group,pirfenidone treated group(positive control),low dose of phlorizin nanoparticles treated group,and high dose of phlorizin nanoparticles treated group.A mouse model of pulmonary fibrosis was established by intratracheal injection of bleomycin solution.Treated administration began 14 days later.Among them,phlorizin nanoparticles are administered through intervaginal space injection(ISI).After 28 days,the mice were killed for sampling.The efficacy was evaluated by histological indicators(Masson stain,Sirius scarlet stain),imaging indicators(computed tomography),non-invasive airway mechanics testing,and biomechanopharmacological indicators(lung-tissue-stretch test,Young’s modulus of lung tissue).Results1.Phlorizin has a significant therapeutic effect on liver fibrosis.It can decrease collagen deposition in liver tissue significantly.It can reduce the content of hyaluronic acid and laminin in the blood,as well as improve liver function in mice with liver fibrosis.The results of ultrasound experiments show that phlorizin can significantly improve the density and hardness of the liver in mice with liver fibrosis.Cell experiments have shown that phlorizin can regulate the morphology of the cytoskeleton of human hepatic stellate cells(LX-2).Those results indicate that phlorizin can regulate the mechanical properties of the liver from micro to macro levels.2.mRNA and LncRNA sequencing,GO functions annotation,KEGG pathway enrichment analysis,interaction relationship with LncRNA-mRNA,and target gene prediction analysis were performed on different groups of liver tissues.The results suggest that there are obvious differences in the mechanism of phlorizin and silibinin in the treatment of liver fibrosis.After molecular docking calculation,it was found that phlorizin had good binding ability to ferroptosis-related proteins,and the binding energy was<-5 kcal/mol.After treated with phlorizin,the iron content in the tissues of liver fibrosis was significantly reduced.These results indicated that the mechanisms of phloridzin treated liver fibrosis is related to the ferroptosis signaling pathway.3.The optimal process for preparing the phlorizin-phospholipid complex is as follows:the reaction solvent is tetrahydrofuran;The drug phosphorus ratio is 1:1;The reaction time is 2 h;The reaction temperature is 40℃.After preparing the complex,wrap the nanoparticles with hyaluronic acid.The particle size and zeta potential of the nanoparticles were measured by using a nanoparticle size analyzer.UV and IR absorption spectroscopy experiments proved that the drug was loaded into the particles.XRD,DSC analysis results showed that the crystal structure of the phlorizin disappeared and was uniformly dispersed in lecithin with an amorphous state.The drug load rate was 31.73±2.87%,and the encapsulation rate was 75.65±3.41%.The in vitro release rate of nanoparticles was 65%at 24 h,more than 75%at 48 h,and complete release at 48 h.4.The treatment of pulmonary fibrosis with prepared phlorizin nanoparticles by ISI showed that this treatment method can effectively reduce the collagen deposition area in the lung tissue,effectively improve the lung function of pulmonary fibrosis mice,increase the flexibility of the lung tissue of pulmonary fibrosis mice,and reduce their Young’s modulus.Conclusion and SignificanceFirstly,we have used phlorizin to treat liver and pulmonary fibrosis and revealed its pharmacological mechanism.This is the first time that these results and mechanisms have been mentioned in the pharmaceutical research of phlorizin.We have also proposed new therapeutic ideas based on energy metabolism,ferroptosis,and regulation of biomechanical related LncRNAs.Secondly,we explore a new type of drug delivery method for the treatment of pulmonary fibrosis,that is,the drug delivery method of nanoparticles combined with ISI.The significance lies in the treatment group’s fibrosis,and also lies in exploring the application prospects of the drug delivery method of nanoparticles combined with ISI.Thirdly,another feature of this study is to verify the efficacy from the perspective of tissue mechanics.The research and standardization of physical parameters for many diseases and drugs is still in a relatively blank field,and it is urgent for researchers to explore.These results and conclusions provide a reference for future research on the treatment of tissue fibrosis.