| Objective:There are two major challenges in the drug treatment of malignant tumors.One is that the drug dosage is too low and the treatment time is too short to lead to unsatisfactory treatment effects;second,most chemotherapy drugs are not targeted,and there are great toxic side effects on the body.And many patients do not die from malignant tumors,but from tissue and organ damage caused by chemotherapy.The liver is an important detoxification and drug metabolism organ of the body,and chemotherapy drugs can easily damage the liver directly or indirectly.So how to alleviate liver damage in the process of chemotherapy deserves our attention.Different chemotherapy drugs have different mechanisms,causing inconsistent damage to the body,and most chemotherapy drugs can cause liver damage,including toxic hepatitis,liver fibrosis,hepatocyte necrosis,hepatocyte steatosis,etc.Clinically,suspected drugs must be stopped immediately when chemotherapy leads to severe liver injury;for mild liver injury,it is necessary to reduce the dosage of chemotherapy drugs,shorten the medication time,and cooperate with anti-inflammatory,antioxidant,detoxification,enzyme reduction,anti-yellowing,and other liver-protective drugs for adjuvant treatment.5-Fluorouracil(5-FU)is a pyrimidine nucleotide synthesis inhibitor,which is metabolized by dihydropyrimidine dehydrogenase in the liver,which can inhibit thymidine synthase,thereby interfering with DNA and RNA synthesis.5-FU belongs to the anti-metabolic chemotherapeutic drugs.5-FU has a good anti-tumor effect,but at the same time it also has serious toxic side effects on the body,and the toxic side effects and mechanisms of the liver are still unclear.Therefore,an in-depth discussion of the toxic side effects of 5-FU on the liver not only has important theoretical significance for explaining its pharmacological mechanism but also has guiding value for finding a protective method for clinical prevention and treatment of liver damage caused by chemotherapy.Angelica sinensis is an important medicine for "tonifying and activating blood" in traditional Chinese medicine,which has been used for thousands of years.Studies have shown that Angelica sinensis has a variety of pharmacological functions,including anti-tumor,antithrombotic,antioxidant,anti-radiation,anti-inflammatory,antibacterial,enhancing immune function,and so on.Angelica polysaccharide(ASP)is an important drug active component of Angelica sinensis.It is reported that ASP can not only promote bone marrow hematopoietic function and "replenish blood",but also inhibit leukemia cell proliferation and promote its differentiation to maturity.It has the effect of "dispelling evil".It can be seen that ASP has the effect of "two-way regulation".Our previous studies confirmed that ASP could antagonize the damage of 5-FU to bone marrow stromal cells,reduce the damage of bone marrow hematopoietic cells and delay premature cell aging.Our latest research shows that ASP could reverse the imbalance of osteogenesis or lipid differentiation induced by 5-FU,suggesting that ASP has an antioxidant effect.However,whether the antioxidant effect of ASP can antagonize the damage of 5-FU to the liver in tumor treatment is worthy of further study.According to the latest oxidative damage theory,the abnormal antioxidant pathway mediated by transcription factor Nrf2 is the key link leading to oxidative stress injury.In this study,the mechanism of ASP regulation of Nrf2/HO-1 signaling pathway and antagonism of 5-FU-induced liver damage is explained by constructing a 5-FU hepatic and hepatocellular injury model.The purpose of this study is to provide a new theoretical basis for explaining the "theory of tonifying and activating blood","theory of supporting righteousness and eliminating evil" and the modern antioxidant theory of traditional Chinese medicine;To provide a laboratory basis for ASP in the prevention and treatment of the liver injury caused by chemotherapeutic drugs;In order to provide effective natural protective agents to reduce the hepatotoxicity caused by 5-FU and provide new targets for the treatment of liver injury.Methods:1.Detection of morphological damage of liver and hepatocytesThe changes of liver tissue structure were observed by HE staining.TUNEL staining was used to detect liver cell apoptosis.PAS staining was used to detect liver glycogen.SA-β-gal staining was used to detect the senescence of liver cells.The change of liver fibrosis was analyzed by Masson staining.Lipid deposition in liver cells was detected by Oil Red O staining.The ultrastructure of liver cells was observed by transmission electron microscope.2.Detection of functional related indexes of liver and hepatocyte injuryThe contents of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in serum and liver were measured by enzymatic method.The contents of triglyceride(TG)and cholesterol(TC)in liver were measured by microplate method.Reactive oxygen species(ROS)staining was used to measure the content of ROS in liver tissue,and DCFH-DA method was used to detect the content of ROS in MIHA cells.The activities of superoxide dismutase(SOD)and lactate catalase(CAT),and the contents of nitric oxide(NO)and malondialdehyde(MDA)in liver were detected by corresponding kits.3.Mechanism analysis of liver and hepatocyte injuryRT-qPCR was used to analyze the expression of genes(Sirt1,Scd1,Ppargc1a,PPAR-γ,PPAR-α,Pnpla2,Mlxipl,Mgat1,Ldlr,G6 PC,Foxo1,Fgf21,Dgat2,Dgat1,Cpt1 a,Cd36,AMPK,and Acadl)related to lipid anabolism;RT-qPCR was used to analyze the expression of endoplasmic reticulum stress-related genes(XBP1,PERK,IRE1α,GRP78,e IF2α,CREB1,CHOP,ATF4,and ATF6);The expression levels of Bcl-2,Bax,Keap1 and HO-1 proteins in liver tissue and MIHA cells were analyzed by Western blot;The level of Keap1 protein in liver tissue was measured by immunohistochemistry;The levels of 8-OHd G and HO-1 protein were detected by immunofluorescence;The nuclear translocation of Nrf2 protein was observed by Western blot and immunofluorescence.Results:1.Morphological test results of liver and hepatocyte injury1)General morphological indicators of liver: The liver of mice in the 5-FU group was dark yellow in color and greasy in appearance,and the liver weight was significantly reduced,with a statistically significant difference(P < 0.01).The color of the liver of mice in the ASP + 5-FU group was redder and the liver weight was significantly increased compared with 5-FU group,suggesting that ASP could antagonize the reduction of liver weight in mice induced by 5-FU(P < 0.05).2)Liver histomorphological: There was no abnormality in the liver tissue of mice in the control group and ASP group,and the hepatocytes were arranged orderly.In the 5-FU group,the hepatocytes were arranged disorderly,and the cell vacuolation and cell swelling were obvious(P < 0.001).Compared with 5-FU group,the damage of liver tissue in ASP + 5-FU group was less severe,the arrangement of hepatocytes was orderly,and the cells had no obvious vacuolation and swelling(P < 0.001).3)Liver fibrosis: Compared with the control group,the collagen fibers in the liver tissue of the 5-FU group were significantly increased(P < 0.001).Compared with the 5-FU group,the collagen fibers in the liver tissue of the ASP + 5-FU group were significantly decreased(P < 0.001).4)Index of liver tissue glycogen content: Compared with the control group,the liver tissue glycogen content in the 5-FU group was significantly decreased(P < 0.001).Compared with the 5-FU group,the glycogen content in the liver tissue of the ASP + 5-FU group was significantly increased(P < 0.05).5)Lipid drop content index of liver tissue: Compared with the control group,the lipid drops in liver tissue and in MIHA cells in the 5-FU group were significantly increased(P < 0.001).Compared with the 5-FU group,the lipid droplets in the liver tissue and in MIHA cells of the ASP+5-FU group were decreased(P < 0.001).6)Detection indicators of liver senescent cells: SA-β-gal staining results showed that there was no significant difference in the number of liver senescent cells among the groups.7)Detection index of the number of apoptotic cells in the liver tissue:TUNEL staining results showed that the number of apoptotic cells in the liver tissue of the 5-FU group increased significantly(P < 0.001).Compared with the 5-FU group,the number of apoptotic cells in the liver tissue of the ASP + 5-FU group was significantly decreased(P < 0.001).8)Observation indicators of ultrastructure of liver tissue and MIHA cells: The results of transmission electron microscope showed that the mitochondria of hepatocytes in 5-FU group were swollen,a large number of lipid droplets were deposited in the cells,and the nuclear volume became smaller and nuclear pyknosis.In ASP + 5-FU group,the swelling of mitochondria in liver tissue cells was reduced,there was no obvious lipid droplet deposition in cells,and the nuclear abnormality was not obvious;The mitochondria and endoplasmic reticulum of MIHA cells in 5-FU group were swollen significantly,and contained a large number of lipid droplets.In ASP + 5-FU group,the swelling of mitochondria and endoplasmic reticulum in MIHA cells was not obvious,and there was no obvious lipid droplet deposition.2.Functional test results for liver and hepatocyte damage1)Test results of ALT and AST in liver tissue and serum: Compared with the control group,the contents of ALT and AST in serum and liver tissue of mice in 5-FU group increased significantly(P < 0.01).Compared with 5-FU group,the contents of ALT and AST in serum and liver tissue of ASP + 5-FU group were significantly decreased.2)The detection results of TC and TG in liver tissue: Compared with the control group,5-FU had no significant effect on the content of TC in liver tissue.However,the content of TG in the liver tissue of mice in the 5-FU group was significantly increased(P < 0.001).Compared with the 5-FU group,the contents of TG in the ASP + 5-FU group was significantly decreased(P < 0.001).3)Detection results of ROS in liver tissues and MIHA cells: Compared with the control group,the content of ROS in liver tissues and MIHA cells of mice in 5-FU group were increased significantly(P < 0.001).Compared with 5-FU group,the content of ROS in liver tissue and MIHA cells of ASP + 5-FU group were decreased significantly(P < 0.001).4)Antioxidant detection results in liver tissue: Compared with the control group,SOD and CAT activities in liver tissue of 5-FU group were significantly decreased(P < 0.05).Compared with the 5-FU group,SOD and CAT activities in the ASP + 5-FU group were significantly increased(P < 0.05).5)The detection results of lipid peroxides in liver tissue: Compared with the control group,the contents of MDA and NO in the liver tissue of the 5-FU group were significantly increased(P < 0.001).Compared with the 5-FU group,the contents of MDA and NO in the liver tissue of the ASP + 5-FU group were significantly decreased(P < 0.001).3.Detection results of liver tissue and hepatocyte injury mechanism1)Detection results of apoptosis-related proteins in liver tissue and MIHA cells:Western blot results showed that compared with the control group,the level of the pro-apoptotic protein Bax in the liver tissue and MIHA cells of the mice in the 5-FU group was significantly increased(P < 0.001),while the level of the anti-apoptotic protein Bcl-2 was significantly decreased.Compared with the 5-FU group,the level of the pro-apoptotic protein Bax in the liver tissue and MIHA cells of the ASP + 5-FU group was significantly decreased(P < 0.01),and the level of the anti-apoptotic protein Bcl-2 was significantly increased(P < 0.05).2)Detection results of genes related to lipid synthesis and metabolism in liver tissue and MIHA cells:RT-qPCR results showed that compared with the control group,the expressions of PPAR-γ,CD36,and Fgf21 in the liver tissue of the mice in the 5-FU group were significantly increased.The expressions of Ppargc1 a,Mlxipl,G6 pc,Foxo1,Pnpla2,AMPK,Acadl,Cpt1 a,Mgat1,and PPAR-α were significantly decreased(P < 0.05).There was no significant difference in the expressions of Sirt1,Scd1,Dgat1,Dgat2,and Ldlr.Compared with the control group,the expressions of CPT1 A,FOXO1,PPARGC1 A,PPAR-α,PNPLA2,Acadl,AMPK,G6 PC,Mgat1,and Mlxipl were significantly decreased in MIHA cells in the 5-FU group;the expressions of CD36,Fgf21 and PPAR-γ were increased significantly.Compared with the 5-FU group,the expressions of CPT1 A,FOXO1,PPARGC1 A,PPAR-α,PNPLA2,Acadl,AMPK,G6 PC,Mgat1,and Mlxipl in MIHA cells and in the ASP + 5-FU group were significantly increased,and the difference was statistically significant.The expressions of CD36,Fgf21,and PPAR-γ were significantly decreased,and the difference was statistically significant.3)Detection results of endoplasmic reticulum stress-related genes in MIHA cells:RT-qPCR results showed that compared with the control group,the expressions of PERK,GRP78,e IF2α,and CREB1 in MIHA cells in the 5-FU group were significantly increased.Compared with the 5-FU group,the expressions of CREB1,CHOP,and ATF4 in MIHA cells in the ASP + 5-FU group were significantly decreased(P < 0.05),while the other gene expression differences were not statistically significant.4)Detection results of DNA damage related protein marker 8-OHd G in MIHA cells:The immunofluorescence results showed that the content of DNA damage marker 8-OHd G in MIHA cells in the 5-FU group was significantly increased compared with the control group(P < 0.001).Compared with the 5-FU group,the 8-OHd G content in MIHA cells in the ASP+5-FU group was significantly decreased(P < 0.001).5)Expression results of Nrf2 signaling pathway-related proteins in mice liver tissue and MIHA cells:Liver tissue of mice: The immunofluorescence results showed that compared with the control group,the level of Nrf2 in the nucleus in the 5-FU group was significantly decreased(P < 0.001).Compared with the 5-FU group,the expression level of Nrf2 in the nucleus in the ASP + 5-FU group was significantly increased(P < 0.05).The results of Western blot were consistent with the results of immunofluorescence.The results of immunohistochemistry showed that compared with the control group,the Keap1 protein level in the 5-FU group was significantly increased(P < 0.01).Compared with the 5-FU group,the Keap1 protein level in the ASP + 5-FU group was decreased,and the difference was statistically significant.The results of immunofluorescence showed that the expression level of HO-1 protein in the 5-FU group was significantly decreased(P < 0.001).Compared with the 5-FU group,the HO-1 protein level was increased in the ASP + 5-FU group(P < 0.001).The results of Western blot were consistent with the above results.MIHA cells: The results of immunofluorescence and Western blot showed that compared with the control group,the level of Nrf2 in the cytoplasm in the 5-FU group was significantly increased,but the level of Nrf2 in the nucleus was significantly decreased(P < 0.001).Compared with the 5-FU group,the level of Nrf2 in the nucleus was significantly increased in the ASP + 5-FU group,but the level of Nrf2 in the cytoplasm was significantly decreased(P < 0.001).Western blot results showed that compared with the control group,the level of Keap1 protein in the 5-FU group was significantly increased(P < 0.001).Compared with the 5-FU group,the Keap1 protein level was decreased in the ASP+5-FU group(P < 0.001).Compared with the control group,the level of HO-1 protein in the 5-FU group was significantly decreased(P < 0.05).Compared with the 5-FU group,the HO-1 protein level was increased in the ASP + 5-FU group(P < 0.001).The results of immunofluorescence detection were consistent with the above results.Conclusion1.5-FU can cause structural and functional damage to liver tissue and liver cells,which can be used for the establishment of liver injury models and the study of their prevention and control mechanisms.2.ASP can antagonize the structural and functional damage of liver tissue and liver cells caused by 5-FU,and can be used as a protective agent for the liver during chemotherapy.3.The reduction of 5-FU-induced liver injury by ASP may be closely related to the activation of Nrf2/HO-1 signaling pathway. |
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