| Research background:A variety of causes,such as environmental pollutants,drug side effects,and viral or fungal infections,are the source of pulmonary fibrosis,a chronic progressive lung disorder.At the same time,pulmonary fibrosis is also considered to be one of the major pulmonary complications of COVID-19 patients.At present,the internationally approved drugs for the treatment of pulmonary fibrosis include Nitanib and Pirfenidone,which can protect the lung function of patients by slowing down the progress of pulmonary fibrosis,but neither of these drugs has the reversibility of fibrosis.Scientists have done a lot of research on the pathogenesis of pulmonary fibrosis,and the research shows that metabolic disorders are related to the pathogenesis of fibrosis in lung,kidney,heart and other tissues.In order to cope with the high energy demand of myofibroblasts,including proliferation and production of extracellular matrix,activated myofibroblasts show enhanced aerobic glycolysis ability.If they can limit the energy supply of fibroblasts and myofibroblasts,they will also prevent or delay the occurrence of fibrosis.However,there is still a lack of methods based on metabolism to treat fibrosis.One of the most plentiful polyphenol components in human nutrition is chlorogenic acid.Research has demonstrated that chlorogenic acid can control lipid and glucose metabolism in diseases related to genetic and health metabolism.In the bleomycin pulmonary fibrosis model,chlorogenic acid can alleviate pulmonary fibrosis by inhibiting endoplasmic reticulum stress pathway.Chlorogenic acid can also regulate TGF-β 1/Smad7 pathway alleviates CCI4-induced liver fibrosis in rats,however,the specific mechanism of chlorogenic acid in anti-fibrosis is still unclear.Research purpose:The protective effect of chlorogenic acid on bleomycin-induced pulmonary fibrosis in elderly mice In our study,the model of pulmonary fibrosis in elderly mice was established by intratracheal injection of bleomycin,and the effect of chlorogenic acid on the expression of glycolytic enzymes PFKP,PFKFB3,HK-II and plasma lactic acid was observed by intraperitoneal injection of chlorogenic acid,so as to explore the mechanism of chlorogenic acid in alleviating the model.Research object and method:1.Randomly dividing 48 aged male mice into four groups,the control group(Contral),bleomycin group(BLM group),low-dose chlorogenic acid group(40mg/kg,LCGA group),and high-dose chlorogenic acid group(80mg/kg,HCGA group),12 each,and fed adaptively for 7 days.Intraperitoneal injection 200 μ L ketamine was used to anesthetize mice.Control group: intratracheal injection of 50 μ L Normal saline,injected intraperitoneally100μ L Normal saline every day,lasting for 3 weeks.Intra-arterial injection of 5 mg/kg of bleomycin at a concentration of 50 μl is the Bleomycin group,intraperitoneal injections per day 100 μ L Normal saline,lasting for 3weeks.Low-dose chlorogenic acid group(40mg/kg): intratracheal injection of 5mg/kg bleomycin 50 μ l.40 mg/kg chlorogenic acid 100μ l per day,lasting for 3weeks.High-dose chlorogenic acid group(80mg/kg): intratracheal injection of 5mg/kg bleomycin 50 μ l.80 mg/kg chlorogenic acid 100μ l per day,it lasts for 3 weeks.2.On day 28,mice are euthanized by intraperitoneal injection of ketamine anesthesia,and lung index = lung wet weight(mg)/ body weight(g)is calculated.From the date of injection of bleomycin,the body weight of the aged mice was measured every 7 days,and the general conditions of the mice(respiration,spirit,diet,exercise)were recorded.HE staining and Masson trichrome staining were used to detect the inflammation and fibrosis of lung tissue,and colorimetric method was used to detect the lactic acid value of mouse plasma.Immunohistochemical staining was used to detect the expression of PFKP,PFKFB3,HK-II proteins,RT-PCR was used to detect the expression of PFKP,PFKFB3,HK-II m RNA,and Western blot was used to detect PFKP,PFKFB3,HK-II α-SMA and Collagen I,Collagen III protein expression.Result:1.General situation of rats: during the experimental recording period on the 7th,14 th,21th and 28 th days,the control group mice were in good mental condition,well developed,normal foraging,smooth breathing,quick response and weight gain.In the bleomycin group,the mental state gradually deteriorated,the number of foraging gradually decreased,the number of dyspnea gradually increased,the reaction became slow,the activity decreased,and the weight gradually decreased.After the intervention of chlorogenic acid,the mental state and foraging of mice were normal,and the response was relatively agile.The weight loss was less than that of the model group as time progressed.The weight loss of mice in the high-dose chlorogenic acid group(80mg/kg,HCGA group)was the lowest.By calculating the lung index,it was found that the lung coefficient of mice in BLM group was significantly higher than that in the control group,and after chlorogenic acid intervention,the lung coefficient of mice in bleomycin group was reduced,and the lung coefficient of mice was reduced by80mg/kg of chlorogenic acid.2.Conditions of isolated lung tissue: After the experiment on the 28 th day,we found that the lung tissue of the control group was very smooth and moist,without any bleeding points or local scars,and had good elasticity.In the bleomycin group,the lung tissue volume of mice was significantly reduced,became hard,the surface was not smooth,and a large number of bleeding spots and scars could be seen.After treatment with chlorogenic acid,the lung tissue elasticity of mice was general and the volume was reduced,but it was between the control group and bleomycin.There were a small amount of bleeding spots and scar depressions on the surface of lung tissue.The overall situation of isolated lung tissue of mice in the 80 mg/kg chlorogenic acid group in the intervention group was close to that in the control group.3.Pathological condition of lung tissue:(1)HE staining showed that the lung tissue structure of the control group mice was complete,no obvious alveolar rupture or inflammatory reaction was found,and no fibrous scar tissue hyperplasia was found.In the mice in the bleomycin group,the alveolar structure was seriously damaged,broken,the interval became wider,the inflammatory cells infiltrated significantly,and the fibrous scar tissue also proliferated.After chlorogenic acid intervention,the destruction of alveolar structure was reduced,inflammatory cell infiltration in lung tissue,alveolar septal thickening and fibrous scar tissue hyperplasia were observed,but the overall improvement of lung inflammatory reaction and destruction of alveolar structure was observed.In the high-dose chlorogenic acid group(80mg/kg),mild alveolar inflammation was observed,and the alveolar structure was close to normal tissue.(2)Masson staining: the alveolar structure of the control group was clear and complete,and there was no blue collagen fibrosis area.In bleomycin group,a large number of blue collagen fibers were deposited,the alveolar structure was seriously damaged,the alveolar septa were significantly thickened,and the fibrosis area was obvious.Compared with the model group,the use of chlorogenic acid can significantly reduce the degree of pulmonary fibrosis after intervention.In the high-dose chlorogenic acid group(80mg/kg),mild pulmonary fibrosis can be observed,and the alveolar structure is closer to normal tissue,which is similar to the HE staining results.4.Szapiel score: by calculating the Szapiel score,compared with the control group,the model group has the highest score,which is significantly different from the control group.The use of chlorogenic acid intervention can improve the lung histopathological score,of which the high dose chlorogenic acid group(80mg/kg)Szapiel score is closer to the normal level.5.Colorimetry: Through the measurement of plasma lactic acid,it was found that the plasma lactic acid level of the bleomycin group was significantly higher than that of the control group,and the measured value of plasma lactic acid was lower than that of the bleomycin group after the intervention of chlorogenic acid,among which the plasma lactic acid level of the 80 mg/kg chlorogenic acid group in the intervention group decreased more significantly than that of the model group.6.Immunohistochemical staining: compared with the control group,the brown positive areas of PFKP,PFKFB3 and HK-II in the bleomycin group were significantly increased,and chlorogenic acid was used(40,80mg/kg/d × 21 days)after treatment and intervention,the expression of PFKP,PFKFB3,and HK-II brown positive areas was lower than that of bleomycin group,and the expression of PFKP,PFKFB3,and HK-II brown positive areas in the high-dose chlorogenic acid group(80mg/kg)decreased significantly compared with the model group.7.RT-PCR results: The m RNA expression of PFKP,PFKFB3 and HK-II in bleomycin group was significantly higher than that in control group.Chlorogenic acid(40,80 mg/kg/d × The m RNA expression of PFKP,PFKFB3 and HK-II after treatment intervention was lower than that of bleomycin group.8.Western blot: Compared with the control group,PFKP,PFKFB3,HK-II α-The protein expression of SMA,Collagen I and Collagen III was significantly increased,and chlorogenic acid was used(40,80mg/kg/d × 21 days)PFKP,PFKFB3,HK-II α-The protein expression of SMA,Collagen I and Collagen III was lower than that of bleomycin group.Conclusion:1.We successfully established a mouse model of pulmonary fibrosis by intratracheal injection of bleomycin.2.Chlorogenic acid has protective effect on pulmonary fibrosis in mice.3.Chlorogenic acid can alleviate bleomycin-induced pulmonary fibrosis by inhibiting glycolysis pathway,which may be the mechanism of alleviating pulmonary fibrosis.Chlorogenic acid and targeted glucose metabolism therapy can provide new therapeutic options for clinical treatment of pulmonary fibrosis. |
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