Protective Effects Of Resveratrol And FGF1 Synergistically Ameliorates On Doxorubicin-induced Acute Cardiotoxicity

BackgroundDoxorubicin(DOX)is a highly effective anthracycline drug,which can be used in the treatment of a variety of malignant tumors.It is the most widely used anti-tumor drug in clinical use.However,due to the abnormal REDOX cycle caused by DOX,its clinical use in cancer therapy is limited by time-and dose-dependent cardiotoxicity,which has caused widespread concern.To date,there is no established definitive or effective treatment.Acute cardiotoxicity due to DOX typically occurs during or within days to weeks of treatment and occurs in approximately 11%of patients.The symptoms of acute cardiotoxicity are mostly reversible.Early identification and intervention of DOX-induced acute cardiotoxicity are essential to reduce adverse reactions,improve the quality of life of patients,and obtain a better prognosis.Exogenous supplementation of fibroblast growth factor 1(FGF1)can reduce DOX-induced cardiomyocyte apoptosis,thereby delaying the degree of myocardial fibrosis and significantly improving left ventricular function.FGF1,a founder member of the FGF family,is known for its antioxidant effects in treating cardiovascular disease and is involved in a wide range of biological activities.However,FGF1 has strong mitogenic activity and long-term use increases the tumorigenic risk,limiting the use of FGF 1 in vivo.How to further exert the cardioprotective effects of FGF 1 while inhibiting its proliferative activity still needs further investigation.The effectiveness of natural products in inhibiting cell proliferation and interfering with tumorigenic signaling pathways is well established.Additionally,natural products have been found to be advantageous in the treatment of cardiovascular diseases.This suggests that we can further exert the cardioprotective effect of FGF 1 by finding a natural product that inhibits its proliferative activity.Resveratrol(RES),a natural polyphenol compound,has attracted considerable attention for its anti-proliferative properties,and can be used as an alternative or adjuvant therapy in combination with other chemotherapy drugs.In addition,RES has anti-inflammatory,antioxidant,antithrombotic,vasodilation,apoptosis inhibition,and other pharmacological effects.Acute treatment with low doses of resveratrol has been shown to have cardio-protective effects in a variety of disease models.RES can not only prevent or slow down the progression of malignant tumors,but also improve the anti-cancer effect of DOX.However,it remains unclear whether RES can enhance the anticancer effects of DOX while simultaneously inhibiting the mitogenic activity of FGF1 and exerting its protective effect against DOX-induced cardiotoxicity.Recent studies have shown that resistance of RES to DOX-induced oxidative stress depends on the activation of sirtuin 1(SIRT1).SIRT1 mediates the protective effect of FGF1 against DOX-induced cardiotoxicity.In addition,down-regulating the expression of nuclear factor erythroid 2-related factor 2(NRF2)can further aggravate DOX-induced myocardial oxidative damage,while RES can increase the expression and transcriptional activity of NRF2 to reduce oxidative stress damage.FGF1 also reduces oxidative stress by stimulating the nuclear translocation of NRF2 and increasing the expression of antioxidant proteins.However,it remains unknown whether RES and FGF1 inhibit cardiotoxicity after DOX therapy by activating the SIRT1/NRF2 pathway.Objectives1.To verify the antagonistic effect of RES on the proliferative activity of FGF1 and the protective effect of the combination of RES and RES on acute cardiotoxicity induced by DOX.2.To explore the mediating effect of NRF2 on the combined treatment of RES and FGF1 to alleviate acute cardiotoxicity caused by DOX.3.To explore whether the protective effect of RES combined with FGF1 on acute cardiotoxicity induced by DOX is dependent on SIRT1,and the regulatory mechanism between SIRT1 and NRF2.Methods1.Construction of animal modelEight-week-old C57BL/6 male mice were randomly divided into five groups of six mice each.Intraperitoneally injected RES(10 mg/kg/day),FGF1(0.5 mg/kg/day)or the same volume of normal saline for 7 days according to the group.After 7 days,each group was injected intraperitoneally with 20 mg/kg DOX or the same volume of normal saline and euthanized after 24 hours.2.Construction of cell modelHepG2,5637,and MCF-7 cells were treated with 20 μM RES and/or 100 ng/ml FGF1.The Nrf2 or Sirt1 gene in H9c2 cells was silenced using the shRNA transfection technique.The cells were then treated with 1μM DOX,20 μM RES,100 ng/ml FGF1 and SFN either separately or simultaneously based on their respective groups for a duration of 24 hours.3.Cell viability testCell viability was measured using CCK-8 kits.4.Apoptosis AssayThe cell apoptosis rate was measured using the TUNEL staining.5.General index detectionContents of CK,LDH,cTnI,MDA and GSH were evaluated.6.Histopathological test6.1 IF stainingIF staining was used to detect the protein expression of CD68,NRF2,HO-1 and NQO1 in the mouse heart.6.2 IHC stainingProtein expression levels of SIRT1 and Cleaved caspase-3,the executor of apoptosis,were measured using IHC staining.7.ROS detectionDHE staining and DCFH-DA staining was utilized to assess the generation of ROS.8.RT-qPCRRT-qPCR was utilized to detect cardiac inflammatory factors in mice:Il1a,Il1b,Il6,Tnfa,and Mcp1 mRNA levels.9.Western blotWestern blot was used to detect both Cleaved caspase-3,BCL-2,BAX,IKBα,p-IKBα,p65,p-p65,CAT,SOD1,SOD2,n-NRF2,HO-1,NQO1,SIRT1,Ac-p53,Ac-FOXO1 and KEAP1.10.Statistical analysisAll data were expressed as mean ± SD.Each group was compared using either a one-way analysis of variance or a two-factor analysis of variance followed by Tukey’s or Sidak’s posterior.P<0.05 was considered to be statistically significant.Results1.RES can significantly antagonize the proliferative activity of FGF1The results of CCK-8 demonstrate that RES effectively inhibits the proliferative activity of FGF1 in HepG2 cells,5637 cells,and MCF-7 cells.TUNEL staining showed that FGF1 therapy inhibited TUNEL-positive cells in MCF-7 cells,a trend that was significantly reversed by RES intervention.2.The combination of RES and FGF1 can reduce acute cardiotoxicity induced by DOX 2.1 The combination of RES and FGF1 can alleviate myocardial injury and apoptosis induced by DOXThe results showed that mice in the DOX group had significantly higher serum levels of CK,LDH and cTnI compared to mice in the Ctrl group.Intervention with RES or FGF1 was able to significantly reduce the above-mentioned myocardial injury index in mice in the DOX group,and the combination of RES and FGF1 further improved the above-mentioned index.IHC staining showed a significant increase in Cleaved caspase-3 protein expression in the DOX group compared to the Ctrl group,and a decrease in DOX-induced Cleaved caspase-3 protein expression in the single administration of RES or FGF1 groups.However,RES in combination with FGF1 further reduces the expression of Cleaved caspase-3 protein.In mice treated with DOX,there was an increase in Cleaved caspase-3 protein expression and a decrease in the BCL-2/BAX ratio compared to the Ctrl group,as shown by western blot analysis.The study found that RES and FGF1 reduced the expression of Cleaved caspase-3 protein and increased the BCL-2/BAX ratio.Additionally,the combination of RES and FGF1 further enhanced these effects.2.2 The combination of RES and FGF1 can relieve cardiac inflammation caused by DOXRT-qPCR results showed a significant increase in mRNA levels of Il1a,Il1b,Il6,Tnfa and Mcp1 in the DOX group.The intervention of RES or FGF1 was able to significantly reduce the above cardiac inflammatory indicators in the DOX group,with the improvement of RES in combination with FGF1 being more significant.IF staining showed that DOX-induced increased expression of CD68 protein in the heart of mice treated with RES and/or FGF1 reduced compared to the Ctrl group.Western blot results showed that the ratios of p-IKBα/IKBα and p-p65/p65 were significantly increased by DOX intervention,but decreased by RES or FGF1 alone.This trend is further suppressed by the combination of RES and FGF1 in the DOX group.2.3 The combination of RES and FGF1 can improve cardiac oxidative stress induced by DOXResults from DHE staining showed that ROS production was significantly increased in the hearts of mice in the DOX group compared to the Ctrl group,and that ROS production was significantly reduced by either RES or FGF1 administration alone,and further reduced by a combination of RES and FGF1 administration.The results of the tests indicated a noteworthy rise in MDA levels and a decline in GSH levels in the mice treated with DOX.However,there was a significant decrease in MDA levels and an increase in GSH levels in the groups treated with RES or FGF1.The combination of RES and FGF1 also suppressed changes in these indices in the DOX group.Western blot results showed that DOX intervention significantly reduced the expression of CAT,SOD1,and SOD2,while RES and/or FGF1 therapy significantly improved the expression of the aforementioned indices compared to the Ctrl group.3.Combined treatment with RES and FGF1 increased the activity of NRF2 in DOX-induced cardiotoxicityIF staining and western blotting results showed significant increases in expression of the DOX-induced n-NRF2 protein,HO-1,and NQO1 proteins in the hearts of mice,and RES or FGF1 alone enhanced the DOX-induced changes in the aforementioned indices.The combined application of RES and FGF1 further enhances its protein expression on this basis.DHE staining and DCFH-DA staining showed that knockout of the Nrf2 gene removed the protective effect of the combination of RES and FGF1 on ROS production induced by DOX.4.The combined treatment of RES and FGF1 increased the activity of NRF2 by activating SIRT1IHC staining results and western blot results showed that the protein expression level of SIRT1 in mouse heart was significantly decreased in DOX group,while the protein expressions of Ac-p53 and Ac-FOXO1 were significantly increased.Treatment with RES and/or FGF1 reversed the above trend.Western blot results showed that Sirt1 knockdown significantly reduced protein expression of SIRT1 and n-NRF2 in H9c2 cells after DOX exposure and significantly increased protein expression of Ac-p53,Ac-FOXO1 and KEAP1 compared to the NC-shRNA group.In addition,the improved effect of the combination of RES and FGF1 on the DOX induced change of the above indices is removed.The results of DHE staining and DCFH-DA staining indicated that the usage of Sirt1-shRNA significantly increased ROS production in H9c2 cells after DOX exposure.Additionally,it eliminated the protective effect of RES and FGF1 combination therapy on oxidative stress damage in H9c2 cells.5.A positive feedback loop was formed between SIRT1 and NRF2 to improve oxidative stress damage caused by DOXAfter transfection with Nrf2-shRNA in H9c2 cells,compared with NC-shRNA group,protein expressions of SIRT1,n-NRF2,HO-1,and NQO1 were significantly decreased in Nrf2 knockdown group,and protein expression of Ac-p53 was increased.Moreover,the reversal of SIRT1,n-NRF2,HO-1,NQO1 and Ac-p53 protein levels under DOX treatment was cancelled by the combined intervention of RES and FGF1.Western blot results showed that the levels of protein expression of SIRT1 and NRF2 were significantly reduced in H9c2 cells treated with DOX after knocking out the Nrf2 gene,while the levels of protein expression of Ac-p53 were significantly increased.The above indexes couldn’t be significantly changed after the addition of SIRT1 agonist RES.After transfection of Sirt1-shRNA in H9c2 cells,the silencing of Sirt1 significantly reduced the protein expression levels of HO-1 and n-NRF2 compared with the NC-shRNA group,and the use of NRF2 agonist SFN did not significantly change the above indexes.Conclusions1.RES can antagonize the proliferative activity of FGF1,and the combined application of both can improve DOX-induced myocardial injury,apoptosis,inflammation and oxidative stress injury.2.The combination of RES and FGF1 can improve oxidative stress damage induced by DOX by activating SIRT1 and thereby increasing NRF2 activity.3.A positive feedback loop is formed between SIRT1 and NRF2 to improve the acute cardiotoxicity induced by DOX.