| Background:Alcoholic liver injury is a dynamic pathological process of liver cell function impairment caused by alcohol.With the aggravation of its pathological change process,alcoholic liver injury can gradually form alcoholic fatty liver,alcoholic hepatitis,alcoholic cirrhosis,and even Alcoholic liver diseases such as hepatocellular carcinoma can lead to more serious clinical outcomes.At present,the molecular mechanism of the occurrence and development of alcoholic liver injury is not fully understood,and there is no clear therapeutic target.From an epidemiological point of view,the prevalence of alcoholic liver disease caused by excessive drinking has obvious regional differences.There is no national statistical data.If there is a research report,the prevalence of alcoholic liver disease in some cities in Liaoning Province is6.10 %,higher than 4.34% in Zhejiang Province.At present,the clinical drugs used to treat alcoholic liver injury mainly include: glucocorticoids,antioxidants,nutritional supplements,traditional Chinese medicines,natural active ingredients,antibody drugs,etc.The cost of drug treatment varies,and the efficacy reports are also controversial.At present,clarifying the pathological process and molecular mechanism of alcoholic liver injury and exploring effective therapeutic drugs are still problems that need to be resolved.Andrographolide is a commonly used traditional Chinese medicine component with anti-inflammatory,antiviral,and immunomodulatory effects in clinical practice.Recent studies have reported that andrographolide may also protect the liver and gallbladder,protect the cardiovascular and cerebrovascular,etc.,suggesting the possible drug effect There are some new effects that are worth developing,which can produce the biological effects of "old medicines and new applications".The author found through preliminary research and clinical observation that andrographolide may have a better effect on the improvement of liver enzymes,but its specific targets have not been clearly elucidated.Therefore,this study firstly described the research status of andrographolide and liver injury using bibliometrics.At the same time,it collected and analyzed the status of andrographolide in a tertiary hospital to study and explore the clinical application of andrographolide;secondly,In this study,a cell model and an animal model of alcoholic liver injury were constructed.Experimental studies on the occurrence and development process and specific pathogenesis of alcoholic liver injury were carried out from both in vitro and in vivo levels.Andrographolide was used to intervene to explore its effects.The protective effect of alcoholic liver injury provides theoretical basis and experimental data for elucidating the pathogenesis of alcoholic liver injury and exploring new clinical treatment drugs.Objective:(1)Through bibliometrics and analysis of the clinical treatment of andrographolide finished drugs in a tertiary hospital,the research hotspots,clinical indications and possible pharmacological mechanism of andrographolide are clarified,in order to explore andrographolide drugs in depth The application in alcoholic liver injury provides a research path.(2)Construct cell models and animal models of alcoholic liver injury,and use andrographolide for intervention to explore the possible mechanism of andrographolide's protective effect on alcoholic liver injury.Methods:(1)Using CNKI,GNBR,Drugbank,and Hetionet databases as data sources,the search subject terms are "andrographolide" and "liver injury" or "Andrographolide" and "Liver Injury",and the search time range is from January 1,2000 to December 31,2020,the document type was a journal document,and the publication year,citation relationship and keywords and other information were extracted from it,and visualized analysis was carried out through the computer code compiled by ourselves.(2)Retrospective analysis of patients who were hospitalized in a tertiary hospital in Jilin Province from January 2015 to December 2019,and were treated with andrographolide injection during the hospital,through analysis of variance,chi-square test,rank sum test and other methods Compare and analyze the changes of blood routine,liver function,renal function,blood sugar,blood lipids and blood ion levels when the patients are admitted and discharged.(3)Select healthy adult male C57BL/6J mice.After anesthesia,they are disinfected with 75% alcohol and put into the ultra-clean workbench.The mice are dissected,the liver is taken out,and the mouse primary hepatocyte suspension is prepared and cultured.After alcohol exposure,low,medium and high doses(0.05,0.1,0.2mmol/L)of andrographolide were used for intervention,and ELISA method was used to detect the content of inflammation-related factors in cell culture fluid,q RT-PCR and Western Blot methods Detect changes in the expression of genes and proteins of inflammation-related signaling pathways in cells.(4)Eight-week-old male C57BL/6J mice,weighing 16-20 g,were randomly divided into blank control group,model group,positive control group,andrographolide low,medium and high dose groups,a total of six groups,each group 15 pieces.The mice in the blank control group were given normal saline by gavage,the mice in the model group were gavage with commercial high liquor for 12 weeks,and the mice in the positive control group were gavage with high liquor and silybin at the same time,the dose was0.1g/kg ·BW·d-1,andrographolide low,medium and high dose groups were given high liquor gavage,and andrographolide 0.05,0.1,0.2g/kg·BW·d-1 were given by gavage at the same time.After modeling,the changes of various biochemical indicators such as serum transaminase,liver tissue lipid metabolism,and anti-oxidation of each group of mice were detected.At the same time,q RT-PCR,Western Blot and other methods were used to detect inflammation-related signal pathway genes in mouse liver tissues.And protein expression.Results:(1)This study uses bibliometrics methods,with "andrographolide" and "liver injury" or "Andrographolide" and "Liver Injury" as the subject headings,collected since 2000,and published in CNKI,GNBR,Drugbank and Hetionet databases The results show that there are 34 studies on andrographolide and liver injury in the CNKI database,and the number of annual publications is gradually increasing.Combined with the results of foreign language databases,it is found that andrographolide and liver injury study mechanism and anti-Related to inflammation signaling pathway.(2)This study included the medical records of patients who used andrographolide injection in a tertiary hospital from January 2015 to December 2019.A total of 207 cases were included.The blood routine,liver function,and liver function of the patients were compared before and after the medication.Changes in renal function,blood sugar,blood lipids and ion levels found that andrographolide improved the patient's blood routine indicators in white blood cells,monocytes,X neutrophils,lymphocytes and liver function indicators in aspartate aminotransferase,prealbumin,etc.It has obvious effects,but the patient's renal function,blood lipids and ion levels did not change significantly before and after the medication,suggesting that andrographolide has certain anti-inflammatory and liver protection effects.(3)Alcohol has a certain toxic effect on mouse primary hepatocytes cultured in vitro,and it is obviously dose-dependent.The IC50 value of the 24 h toxic effect of alcohol on primary mouse hepatocytes cultured in vitro is about 400 mmol/L,and alcohol can cause inflammation-related factors IL-1?,IL-4,and IL-in primary mouse hepatocytes.5.The expression levels of IL-6,NF-?B,TNF-? and CYP2E1 increased significantly,and the intervention of different concentrations of andrographolide showed a protective effect on liver cells,especially in the high-dose(0.2mmol/L)group The effect is the most significant.(4)In this study,an animal model of alcoholic liver injury in mice was successfully established.8 weeks old male C57BL/6J mice were given 4mg/d·kg·BW high liquor for one week and then transitioned to 6mg/d·kg·BW by gavage.After 12 weeks,mice can be induced to increase liver weight and liver index,and the levels of serum transaminase related indexes AST,ALT and ?-GT increased significantly(P<0.05);liver tissue oxidative stress index MDA increased significantly,SOD and GSH decreased significantly(P<0.05);the accumulation of total cholesterol(TC),triglycerides(TG)and ROS in the liver tissues showed that the mice had obvious liver tissue damage after 12 weeks of gavage with high liquor.(5)Andrographolide has obvious protective effect on alcoholic liver injury in mice.The alcohol model group mice were given different concentrations of andrographolide(0.05,0.1,0.2g/kg·BW),and the results showed that the high-dose andrographolide(0.2g/kg·BW)group can significantly reduce the liver of mice The difference in weight and liver index was statistically significant(P<0.05);and the serum aminotransferase AST,ALT and ?-GT levels of mice in the high-dose andrographolide group were significantly lower than those in the alcohol model group,and compared with the blank control group and the positive control There was no significant statistical difference between the two groups;the level of lipid peroxidation index MDA in the liver tissue of mice in the middle and high dose of andrographolide group was significantly lower than that of the alcohol model group,and the difference was statistically significant(P<0.05).The antioxidant index SOD and The level of GSH in the high-dose andrographolide group was significantly higher than that of the model group,and was not significantly different from the positive control group;andrographolide can effectively reduce alcohol-induced lipid disorders in liver tissue to a certain extent,and the high-dose group,ie 0.2 The protective effect of g/kg·BW andrographolide was the most obvious.Compared with the positive control group,the levels of TC and TG in the liver tissue of the high-dose andrographolide group were not statistically different(P>0.05);compared with the model group In comparison,ROS in the liver of mice in each dose group of andrographolide decreased significantly,especially in the middle and high-dose groups,and the difference was statistically significant(P<0.05).The high-dose group had the most obvious effect.And the level of ROS in the liver tissue of mice in the high-dose andrographolide group was not significantly different from that in the positive control group(P>0.05).(6)From the results of liver histopathology(HE staining)in each group of mice,it can be seen that the size of the liver cells of the blank control group and the positive control group is the same,and the hepatocyte cords are arranged radially with the central lobular artery as the center;The nucleus is round and the cytoplasm is evenly red stained;the bile canaliculus cell structure is intact and no disease is seen.Compared with the mice in the blank control group,the liver tissue structure of the mice in the model group was disordered,the liver lobules and hepatic cord structures disappeared,a large number of fatty vacuoles and inflammatory cells were infiltrated,and the cytoplasm of liver cells was sparse,showing fibrosis.Compared with the model group,the liver tissue morphology of mice in each dose of andrographolide has improved,especially in the high-dose(0.2g/kg·BW)group.The liver tissue is close to the structure of the positive control group.Vacuoles,inflammatory cell infiltration and fibrosis were significantly improved.(7)Andrographolide has a significant effect on the expression of NF-?B,TNF-?and CYP2E1 protein levels in the liver tissues of mice in each group.From the results of immunohistochemistry,there is a large amount of NF-?B in the liver tissues of the model group.And TNF-? in situ expression.Compared with the model group,with the increase of the intervention dose,the accumulation of NF-?B and TNF-? in each dose group of andrographolide gradually decreased,and showed a significant dose dependence.The high dose of andrographolide group mice liver tissue NF The expression levels of-?B and TNF-? are close to the levels of the blank control group and the positive control group.From the results of Western Blot,the expression levels of NF-?B,TNF-? and CYP2E1 in the liver tissues of the model group were significantly increased(P<0.05),and after the intervention of andrographolide,NF-?B,TNF-? and The expression level of CYP2E1 decreased,and the difference was statistically significant(P<0.05).The expression levels of NF-?B,TNF-? and CYP2E1 in the andrographolide groups were not significantly different from those in the positive control group(P>0.05).Conclusions:(1)According to the bibliometric analysis,the possible signal pathways of andrographolide and liver injury related research are mainly inflammatory signal molecules,such as NF-?B p100,NF-?B p105,TNF-? and so on.(2)Through the analysis of the current clinical application of andrographolide,it is concluded that andrographolide is a commonly used anti-inflammatory drug in clinical practice and has a certain effect on improving liver function.(3)Alcohol has a certain toxic effect on primary hepatocytes of mice cultured in vitro,showing an obvious dose-dependence.The high-dose andrographolide group(0.2 mmol/L)has obvious effects on primary hepatocyte damage caused by alcohol The protective effect.(4)This study successfully established an animal model of alcoholic liver injury in mice,that is,4 mg/d·kg·BW high liquor was given to 6mg/d·kg·BW by gavage for a total of 12 weeks,which can induce serum Increased transaminase,liver lipid peroxidation,ROS generation and other liver function damage and histopathological changes.(5)The high-dose andrographolide group(0.2g/kg·BW)can effectively reduce serum transaminase levels,oxidative stress levels,liver lipid levels and ROS production in mice with alcoholic liver injury.(6)The protective mechanism of andrographolide on alcoholic liver injury may be achieved by inhibiting the expression of related proteins in the CYP2E1/ROS/NF-?B signaling pathway. |
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