| Background: Asthma is a common chronic airway disease characterized by airway hyperresponsiveness,airway inflammation,and airway remodeling.For most asthma patients,glucocorticoids can control the disease well,but the chronic airflow limitation phenotype is not sensitive to glucocorticoid therapy.Therefore,exploring new targets and therapeutic drugs for asthma remains an unsolved problem.Glutamate-histidyl-L-lysine(glycyl-L-histidyl-L-lysine,GHK)is a naturally occurring tripeptide in human blood,and human plasma GHK levels decrease with age.The ability of GHK and GHK-Cu to participate in tissue repair and anti-inflammatory and antioxidant activities has been confirmed by many studies.After combining with ionic copper in blood,it exerts its biological effect in the form of GHK-Cu complex.The antioxidant activity of GHK-Cu is significantly stronger than that of GHK,and its polypeptide powder is easier to store.A previous drug development report based on gene expression data combined with a Connectivity Map compound analysis predicted that in some chronic airway diseases,such as chronic obstructive pulmonary disease,decreased levels of GHK are associated with disease onset and disease related to severity.Several studies have demonstrated that GHK-Cu has a significant inhibitory effect on the secretion of TGF-β1 in the lung,and our previous studies have also demonstrated that administration of exogenous GHK-Cu can inhibit the EMT mediated by the TGF-β1/Smad signaling pathway.to inhibit bleomycin-induced pulmonary fibrosis in mice.Objective: To determine the correlation between plasma GHK levels and disease severity in asthma patients;to determine whether exogenous supplementation of GHK-Cu can improve airway inflammation and airway remodeling in OVA-induced mouse asthma models,and to explore the effect of GHK-Cu on small Molecular mechanisms related to the improvement of a murine asthma model.Research methods: This study is divided into three parts.The first part investigates the correlation between plasma GHK levels and their lung function in asthma patients,and uses different concentrations of GHK-Cu to intervene in OVA-induced mouse airway remodeling models to evaluate GHK-Therapeutic effects and mechanisms of Cu in a mouse model of airway remodeling.The second part of the study screened the target ofGHK-Cu and verified it in the OVA-induced mouse airway remodeling model.The third part studies the therapeutic effect and mechanism of GHK-Cu in OVA-induced mouse airway inflammation model.1.Correlation between plasma GHK levels and lung function in asthma patients and the therapeutic effect and mechanism of GHK-Cu in OVA-induced mouse airway remodeling model:1.1 Bronchodilatory test: Assess lung function in asthmatic patients.1.2 High-performance liquid chromatography: Assess plasma GHK levels in asthmatic patients.1.3 Grouping of experimental animals and preparation of mouse asthma airway remodeling model: BALB/c wild-type female mice aged 6-8 weeks were randomly divided into 4 groups(n=6)by random number table method,namely the control group(CON group),model group(OVA group),low-dose GHK-Cu(OVA+ 0.2mg/kg GHK-Cu)group(L-GHK-Cu group),high-dose GHK-Cu group(OVA+ 20mg/kg GHK-Cu)Cu)group(H-GHK-Cu group).Except for the control group,other groups were sensitized by intraperitoneal injection of 2ml OVA sensitization solution on the 0th and 14 th days,and the control group was replaced by intraperitoneal injection of the same amount of normal saline.From the 24 th day,the model group and the low-dose and medium-dose GHK groups were given OVA challenge solution by airway inhalation for 18 consecutive days,once a day for 30 minutes per challenge;1 hour before each atomization inhalation of OVA challenge solution,the GHK-Cu group was given the corresponding dose of GHK-Cu treatment solution by intraperitoneal injection,and the model group was given the same volume of normal saline by intraperitoneal injection.The control group was given the same amount of normal saline instead of aerosol challenge and administration at the corresponding time points.1.4 Pulmonary function challenge test: within 48-24 h after the last challenge,use a gradient concentration of methacholine solution to conduct a bronchial challenge test in mice to induce airway hyperresponsiveness,and use whole body plethysmography to evaluate non-invasive lung function in small animals.Changes in airway resistance in mice were assessed using Pehn.1.5 Preparation and staining of paraffin sections of lung tissue: The left lungs of micewere collected,embedded in paraffin and sectioned.HE staining was performed on lung tissue sections to assess inflammatory cell infiltration around the airways;PAS staining was performed on lung tissue sections to assess goblet cell metaplasia in the small airway epithelium;Masson staining was performed on lung tissue sections to assess small airway subepithelial fibrosis condition.1.6 Total count and differential count of bronchoalveolar lavage fluid(BALF)cells:Collect mouse BALF precipitated cells,resuspend and perform cell counting and Giemsa staining,and count the total number of BALF cells and eosinophil count in mice.1.7 Immunohistochemical staining: Immunohistochemical staining was performed on mouse lung tissue sections to evaluate the expression of mouse epithelial-mesenchymal(EMT)phenomenon-related proteins(α-SMA,E-Cadherin and Vimentin)and TGF-β1proteins.1.8 Western Blot: Extract mouse lung tissue proteins and evaluate the expression levels of TGF-β1,smad3 and p-Smad3 protein in mouse lung tissue.2.Screening the target of GHK-Cu and verifying it in the OVA-induced mouse airway remodeling model:2.1 Protein interaction analysis: Based on the biological information database,the target proteins corresponding to GHK were screened,and a protein interaction network was constructed to select the core targets of GHK.2.2 Molecular docking: Calculate whether the selected core target can directly bind to GHK-Cu.2.3 Experimental animal grouping and preparation of asthmatic mouse airway remodeling model: 6-8 weeks BALB/c wild-type female mice were randomly divided into 4 groups(n=6)by random number table method,namely the control group(CON group),model group(OVA group),high-dose GHK-Cu group(OVA+ 20mg/kg GHK-Cu)group(GHK-Cu group),SIRT1 inhibitor(OVA+ 20mg/kg GHK-Cu+ 5mg/kg EX-527)group(EX-527 group).The EX-527 group was given 20mg/kg GHK-Cu treatment solution and 5mg/kg EX-527 intraperitoneal injection.The modeling method is the same as the first part.2.4 Cell treatment and grouping: Human bronchial epithelial-like cells(16HBE)were routinely subcultured,using RPMI-1640 medium containing 10% fetal bovine serum,and cultured under the culture conditions of 5% CO2 and 37 °C.Divided into blank group(GHK-Cu-/HDM-),GHK-Cu control group(10μm GHK-Cu+/ HDM-),HDM group(GHK-Cu-/ HDM +),low-dose GHK group(5μm GHK-Cu+/ HDM+),the high-dose GHK-Cu group(10 μm GHK-Cu+/ HDM +).16 HBE cells were stimulated with corresponding HDM and/or GHK-Cu,respectively.2.5 Determination of SIRT1 deacetylase activity: extract the cell proteins of each group,and measure the SIRT1 deacetylase activity in cells.2.6 Preparation of lung tissue paraffin sections and Masson staining: The left lungs of mice were collected,embedded in paraffin and sectioned.Masson staining was performed on lung tissue sections to assess small airway subepithelial fibrosis.2.7 Immunofluorescence: Detection of SIRT1 expression in mouse airway epithelium.2.8 Immunohistochemistry: To evaluate the expression of epithelial-mesenchymal(EMT)phenomenon-related protein(α-SMA)in each group of mice.2.9 Western Blot: Extraction of mouse lung tissue protein to evaluate the expression level of TGF-β1 protein in mouse lung tissue.3.Therapeutic effect and mechanism of GHK-Cu in OVA-induced mouse airway inflammation model:3.1 Experimental animal grouping and preparation of asthmatic mouse airway inflammation model: BALB/c wild-type female mice aged 6-8 weeks were randomly divided into 5 groups(n=6),namely the control group(NS group),model group(OVA group),low-dose GHK-Cu(OVA+ 0.2mg/kg GHK-Cu)group(L-GHK-Cu group),high-dose GHK-Cu group(OVA+ 20mg/kg GHK-Cu group))group(H-GHK-Cu group),dexamethasone(2 mg/kg)treatment group(DEXA group).Except for the control group,other groups were sensitized by intraperitoneal injection of 2ml OVA sensitization solution on the 0th,7th and 14 th days,and the control group was replaced by intraperitoneal injection of the same amount of normal saline.From the 21 st day,the model group,the low-dose and high-dose GHK groups,and the dexamethasone treatment group were given 5% OVA solution through airway inhalation for 4 consecutive days,once a day,and each challenge was 30 minutes;One hour before the inhalation of OVA challenge solution,the GHK-Cu group was given the corresponding dose of GHK-Cu treatment solution by intraperitoneal injection,the dexamethasone treatment group wasgiven 2 mg/kg dexamethasone solution by intraperitoneal injection,and the model group was given the same amount Intraperitoneal injection of normal saline.The control group was given the same amount of normal saline instead of aerosol challenge and administration at the corresponding time points.3.2 Pulmonary function challenge test: within 48-24 h after the last challenge,use a gradient concentration of methacholine solution to conduct a bronchial challenge test in mice to induce airway hyperresponsiveness,and use whole body plethysmography to evaluate non-invasive lung function in small animals.Changes in airway resistance in mice were assessed using Pehn.3.3 Preparation and staining of paraffin sections of lung tissue: The left lungs of mice were collected,embedded in paraffin and sectioned.Lung tissue sections were stained with HE to evaluate inflammatory cell infiltration around the airways;PAS staining was performed on lung tissue sections to evaluate goblet cell metaplasia in small airway epithelium.3.4 Bronchoalveolar lavage fluid total cell count and differential count: Collect mouse BALF precipitated cells,resuspend them,and perform cell count and Giemsa staining to count the total number of BALF cells and eosinophil count in mice.3.5 ELISA: Collect mouse BALF supernatant to assess the levels of IL-4,IL-13 and TNF-α in BALF3.6 Detection of MDA content: Collect mouse BALF supernatant to evaluate MDA level in BALF3.7 Western Blot: Extract lung tissue proteins and evaluate the expression of Akt,P38,p-Akt and p-P38 proteins in lung tissueResult:1.Correlation between plasma GHK levels and lung function in asthma patients and the therapeutic effect and mechanism of GHK-Cu in OVA-induced mouse airway remodeling model1.1 Both pre-diastolic FEV1 and post-diastolic FEV1 were positively correlated with plasma GHK levels(r=0.566,p=0.011;r=0.530,p=0.020).Pre-diastolic MMEF75/25 also had a moderate positive correlation with plasma GHK levels(r=0.549,p=0.015).1.2 When the concentration of aerosolized methacholine was 50 mg/ml,the Pehn of thelow-dose GHK-Cu group and the high-dose GHK-Cu group were significantly lower than those of the OVA group.1.3 Compared with the OVA group,the infiltration of inflammatory cells around the airways in mice after low-dose and high-dose GHK-Cu intervention was significantly reduced,and the inflammation scores were significantly reduced.1.4 Compared with the OVA group,the total number of inflammatory cells in the BALF of the mice in the low-dose and high-dose GHK-Cu groups were significantly lower,and the number of eosinophils was significantly lower than that in the OVA group.1.5 The mucus secretion and goblet cell metaplasia of mice in the low-dose and high-dose GHK-Cu groups were significantly lower than those in the OVA group.1.6 Compared with the OVA group,the collagen deposition around the airway of the mice in the low-dose and high-dose GHK-Cu groups was significantly reduced.1.7 Compared with the OVA group,the interstitial protein indexes α-SMA and Vimentin around the airway of the mice in the high-dose GHK-Cu group were significantly down-regulated,and the airway epithelial cell junction protein E-Cadherin also showed a significant decrease.increase.This indicated that high-dose GHK-Cu could inhibit the EMT phenomenon in the airway of asthmatic mice.1.8 The immunohistochemical staining results of lung tissue showed that compared with the control group,the expression of TGF-β1 around the airway in the OVA group was increased.1.9 Western Blot results showed that compared with the OVA group,the expression level of TGF-β1 in asthmatic mice treated with high-dose GHK-Cu was down-regulated,the expression level of Smad3 did not change significantly,and the phosphorylation level of Smad3 was significantly down-regulated.This indicated that high-dose GHK-Cu significantly inhibited the TGF-β1/smad3 signaling pathway in asthmatic mice.2.Screening the target of GHK-Cu and verifying it in the OVA-induced mouse airway remodeling model:2.1 The protein interaction network results showed that the targets of GHK were angiotensin II type 1 receptor(AGTR1),adrenergic beta receptor kinase 1(adrenergic beta receptor kinase 1,ADRBK1),silencing Information regulator 1(sirtuin-1,SIRT1).By reviewing asthma-related literature,SIRT1 was selected for subsequent analysis andvalidation.2.2 The simulation results of molecular docking showed that GHK-Cu could directly combine with SIRT1 to form a protein complex,and the interaction sites of the complex consisted of GLU-416,GLU-410,LYS-377 and THR-368.2.3 HDM treatment decreased SIRT1 levels in 16 HBE cells(p < 0.01),while GHK-Cu retraced SIRT1 levels in HDM-stimulated 16 HBE cells in a concentration-dependent manner.2.4 SIRT1 deacetylase activity assay results showed that HDM inhibited SIRT1 deacetylase activity in 16 HBE cells,and GHK-Cu dose-dependently up-regulated SIRT1 deacetylase activity in OVA-stimulated cells.2.5 Immunofluorescence results showed that GHK-Cu intervention could up-regulate SIRT1 levels in OVA-induced asthma mice.2.6 The collagen deposition area around the airways of mice in the GHK-Cu group was significantly lower than that in the OVA group,and this improved effect of GHK-Cu was offset by the additional administration of EX-527.2.7 The results of immunohistochemical staining showed that the expression of α-SMA in the subairway epithelium of mice in the GHK-Cu group was significantly lower than that in the OVA group,and this down-regulation effect of GHK-Cu was offset by the additional administration of EX-527.2.8 Western Blot results showed that compared with the OVA group,GHK-Cu intervention inhibited the expression of TGF-β1 in mouse lung tissue(p<0.001),and the inhibitory effect of GHK-Cu was offset by the additional administration of EX-527.3.Therapeutic effect and mechanism of GHK-Cu in OVA-induced mouse airway inflammation model:3.1 When the concentration of aerosolized methacholine was 50 mg/ml,the Pehn of the low-dose GHK-Cu group and the high-dose GHK-Cu group were significantly lower than those of the OVA group,and the high-dose GHK-Cu had a higher airway response.The improvement effect was similar to the dexamethasone group.3.2 Compared with the OVA group,the goblet cell metaplasia of mice in the high-dose GHK-Cu group was significantly reduced3.3 Compared with the OVA group,the infiltration of inflammatory cells around theairways in mice after low-dose and high-dose GHK-Cu intervention was significantly reduced,and the inflammation scores were significantly reduced,and the therapeutic effect of high-dose GHK-Cu was comparable to that of dexamethasone.There was no statistical difference between the groups.3.4 Compared with the OVA group,the total number of inflammatory cells and the number of eosinophils in the BALF of the mice in the high-dose GHK-Cu group were significantly reduced.There was no significant difference in the total number of inflammatory cells and eosinophils in the BALF between the high-dose GHK-Cu group and the dexamethasone group.3.5 ELISA results showed that compared with the OVA group,both low-dose and high-dose GHK-Cu interventions could reduce the levels of IL-4,IL-13 and TNF-α in the BALF of asthmatic mice.3.6 Compared with the OVA group,both low-dose and high-dose GHK-Cu interventions can reduce the MDA content of BALF.3.7 Western Blot results showed that compared with the OVA group,the phosphorylation levels of Akt and P38 in the lung tissue of asthmatic mice in the low-dose and high-dose GHK-Cu groups were significantly down-regulated.Conclusion:1.Plasma GHK levels in asthma patients are correlated with pulmonary function indicators.2.GHK-Cu can improve airway inflammation and airway remodeling in asthmatic mice.3.GHK-Cu improves airway inflammation in asthmatic mice by inhibiting the level of airway oxidative stress in mice by down-regulating the Akt/MAPK pathway.4.GHK-Cu upregulates the expression level of SIRT1 in mouse airway epithelial cells,and directly binds to SIRT1 and activates its activity5.GHK-Cu inhibits the airway epithelial-mesenchymal transition(EMT)in mice by binding to SIRT1 produced by airway epithelial cells and downregulates the TGF-β1/Smad pathway,thereby improving airway remodeling in asthmatic mice... |
PDF Full Text Request