The Protective Effects And The Possible Mechanism Of Astaxanthin On Liver Injuries Caused By Niu Huang Jie Du Pian In Mice

Objective:Arsenic is a relatively abundance of metalloid elements.Long-term arsenic exposure can lead to parenchymal organ tumors such as lung,liver and spleen,and it is also related to the increased risk of some chronic diseases.According to statistics,about 200 million people around the world are suffering from the poisoning health risks of arsenic.As arsenic has a certain pharmacological effect,it is widely used in the medical field,taking arsenic-containing drugs is also one of the important ways for arsenic exposure in the population.Realgar is the most common arsenic-containing mineral traditional Chinese medicine.Its medicinal value was first seen in Shennong's Materia Medica.It has a long history of medicinal use.Long-term use of realgar-containing drugs in the human body may have elevated serum ALT and AST activity,drug-induced liver disease and cirrhosis and other adverse reactions.NHJDP is a common realgar-containing drug with a wide range of indications,low prices,good efficacy,and a wide patient population.However,due to the significant level of realgar in the dosage form,realgar contains potentially toxic elements such as arsenic,which makes long-term medications.The patients are prone to various arsenic-related health hazards,and chronic arsenic poisoning caused by long-term use of NHJDP has been reported in recent years,of which liver injury is one of the more serious adverse reactions.Astaxanthin is a product of carotenoids widely found in nature and is believed to have dual effects of anti-inflammatory and anti-oxidation.Studies have shown that astaxanthin can play an important role in liver protection by down-regulating the ALT,AST levels in serum,inducing expression of antioxidant enzymes,resisting hepatic fibrosis,and inhibiting inflammatory responses.In this study,animal model was used to induce hepatic injury model by intragastric administration of NHJDP,and astaxanthin was also given to intervene to observe the protective effect and possible mechanism of astaxanthin intervention on liver injury induced by NHJDP.The experimental results will provide experimental basis for using natural antioxidants to prevent and treat drug-induced hepatic injury such as realgar.Methods:1.Experimental animals and grouping:Healthy female Kunming mice,24,weighing 18-20 g?6-8 weeks old?.During the rearing period,the temperature of animal room was strictly controlled at 22?;the relative humidity was maintained at about 55%;and the lighting time was maintained at?12±2?hours per day.After one week of adaptive feeding,the experimental animals were randomly divided into groups of 6 in each group,a total of 4 groups.The groups were:control group,NHJDP exposure group,low-dose ASX intervention group,and high-dose ASX intervention group.Intragastric administration was given twice a day.At 9:00 am,NHJDP was given by gavage,and ASX was given by gavage at 17 points with an interval of 8 h for a total of 6 weeks.The end point of poisoning,CO₂ anesthesia,blood?whole blood or serum?,urine?12 h?and intact liver tissue samples were collected.2.The detection indicators and methods:?1?ALT,AST activity in serum and SOD,GSH content in liver:Nanjing jiancheng kit;?2?Liver histopathological changes:HE staining;?4?Liver MDA content:Shanghai Biyuntian kit;?5?Whole blood GSH content:DTNB method;?6?Liver inflammatory cytokines TNF-?,IL-6,IL-1?,TGF-?1 and oxidative stress related genes m RNA detection of GR,GSTO1/2,NQO1,GCLc and GCLm:Detection by RT-PCR;?6?Expression of MAPK family and its downstream transcription factor NF-?B;Nrf2 signaling pathway and its downstream target gene in the liver:Western Blot method;?7?Detection of arsenic in urine:Atomic absorption method;3.Statistical analysis:Experimental data was analyzed using SPSS 19.0 software and expressed as Mean±SD.ANOVA was used to compare the data between the multiple groups.The SNK method was used to compare the data with each other?the above test levels were all p<0.05?.Results:1.Protective effect of astaxanthin on liver function in oral exposure of NHJDP in miceDuring the administration period,the weight of mice in each group increased steadily.There was no significant difference in body weight and organ coefficient between groups;ASX significantly reduced the increase of ALT and AST activity in serum of mice induced by NHJDP;The liver tissues structure in control group,low dose of ASX and high-dose ASX intervention are normal,and had clear hepatocyte cord structure,normal staining of nuclei and cytoplasm;but in NHJDP group,deep staining of inflammatory cell mass in the central venous are found,indicating inflammatory injury occurred.2.Protective effect of astaxanthin on inflammatory injury on liver of oral exposure of NHJDP in mice.The study found that ASX can significantly inhibit the m RNA expression levels of inflammation-related cytokines TNF-?,IL-6,and IL-1?in the liver tissue of mice exposed to NHJDP and the proteins of the MAPK signaling pathway and the expression level of its downstream transcription factor NF-?B.3.Protective effects of astaxanthin on liver oxidative damage induced by NHJDP oral exposure in mice.ASX intervention significantly inhibited the content of MDA in liver tissues of mice exposed to NHJDP,enhanced SOD activity and up-regulated the expression of GSH in liver tissues and whole blood.ASX can upregulate mRNA and protein expression levels of downstream oxidative stress-related genes GSTO1/2 and NQO1through activation of Nrf2 signaling pathway.4.Effect of astaxanthin on arsenic metabolism and arsenic methylation pattern in oral exposure of NHJDP in miceASX intervention can promote the content of i As,DMA and tAs in urine of NHJDP-disease mice,increase DMA%and SMR,and promote arsenic metabolism and arsenic excretion.Conclusion:1.ASX intervention had a protective effect on liver function impairment in mice induced by NHJDP.2.ASX intervention may inhibit the activation of the MAPK signaling pathway and the downstream transcription factor NF-?B in the liver of mice,and inhibit the inflammatory lesions in the liver of mice induced by NHJDP.3.The ASX intervention may inhibit the oxidative damage in the liver of mice induced by NHJDP by inducing the expression of Nrf2 signaling pathway and its downstream related antioxidant enzymes.4.ASX intervention promoted the metabolism and excretion of total arsenic and various forms of arsenic in NHJDP-exposed mice.