| Objective:Realgar is a Traditional Chinese medicine containing arsenic.Chronic arsenism is often caused by irrational use of Niuhuang Jiedu pills(tablets)containing realgar in China.Its clinical manifestation is systemic damage and the central nervous system is one of the target organs of arsenic toxicity.Niuhuang Jiedu tablets have been banned because of their high arsenic content,and some people have questioned and even opposed the use of realgar as a medicine abroad.The toxic effect of the irrational use of realgar on the human nervous system,have become the focus of attention.In the clinic,Niuhuang Jiedu tablets are a representative drug for the treatment of gastrointestinal heat retention syndrome(GHRS).The main clinical manifestations of GHRS include fever aversion,dry stool,yellow urine,red tongue,etc.Studies have shown that GHRS is related to inflammatory damage caused by imbalance in immune factor secretion and dry stool caused by imbalance in intestinal flora.Intestinal microflora can affect nervous system and intestinal function in many ways,also affect the stress of immune system and the secretion of immune substances.In the state of constipation model,the imbalance of intestinal microbiota is the key regulatory factor to start the brain nerve inflammation,which can participate in the central nerve cell damage through the microbiota-gut-brain axis(MGB)axis,and then lead to cognitive dysfunction.Recent studies have shown that when toxic drugs are used to treat targeted diseases,they show low toxicity or therapeutic effect.Therefore,it is speculated that realgar has a two-way effect of "treatment" and "toxicity" on the central nervous system in pathological state and normal state.On one hand,realgar can make the immune function and intestinal microbiota function of GHRS return to normal,and play its therapeutic role.At the same time,realgar can indirectly correct the imbalance of the key regulatory factors of brain inflammatory response caused by GHRS,which is non-toxic or low toxic to the central nervous system,that is,protective effect.On the other hand,realgar can start the inflammatory reaction of nerve cells in normal body,which shows central nervous system toxicity.MGB axis plays an important role in the regulation of gastrointestinal function and brain health.In this study,metabonomics and microbiomics were used to study the mechanism of MGB axis in the protection and toxicity of realgar on the central nervous system of GHRS rats and normal rats,and then the sensitive molecular targets are identified.This study will provide a theoretical basis and experimental data for re-evaluation of the toxicology and pharmacology of realgar and clinical application value of Niuhuang Jiedu tablets.Methods: 1.Establishment and optimization of animal model of GHRS 80 female Sprague Dawley(SD)rats,3 weeks old,were selected to build a rat model of GHRS with loperamide,high protein and calorie diet.60 rats were randomly divided into ten groups: group Control and group 1?9.An experiment of orthogonal design(L9(34))was used for optimizing the three main factors,the duration of high protein and calorie diet(4 w,8 w,12 w),dosage of loperamide(3 mg/kg,6 mg/kg,12 mg/kg)and the duration of loperamide(5 d,10 d,15 d).The color of tongue and urine were analyzed by photo analysis system;the anal temperature of rats was measured by electronic thermometer;the fecal characteristics were analyzed by Bristol score;the frequency of bowel sounds was recorded by stethoscope;the cold and hot tendency was analyzed by hot and cold stabbing instrument.The remaining 20 rats were divided into 2 groups: model group and control group.Then the experiments above-mentioned was carried out.2.Validation of animal model of GHRS Female SD rats,3 weeks old,were selected and divided into control group,GHRS group,Niuhuangjiedu tablets low-dose and high-dose groups.The changes of tongue,anal temperature,feces and urine were detected by the above methods.The change of ear swelling rate was measured by applying xylene on the ear piece.3.A study of the role of microbiota-gut-brain axis in the protection and toxicity of realgar on GHRS and normal rat hippocampus based on metabonomics and microbiomics Female SD rats,3 weeks old,were selected and divided into control group,1.8g/kg realgar group,GHRS+0.3g/kg realgar group,GHRS+1.8g/kg realgar group,TREAT group.The metabolites in rat hippocampus were determined by liquid chromatography tandem mass spectrometry with no target.16 S r RNA sequencing technology was used to determine the changes of fecal microbial spectrum.Cluster analysis,multivariate statistical analysis,metabolic pathway analysis,correlation analysis,species classification analysis,? and ? diversity analysis,species difference analysis and other bioinformatics methods were used for omics analysis.Arsenic in rat hippocampus were determined by Atomic Fluorescence Spectrometry.The changes of IL-6,NF-?B p65,Iba-1,claudin-5,occludin and MMP-9 in hippocampus were detected by immunohistochemistry and immunohistochemistry.Changes of inflammatory factors in rat serum were determined by ELISA.Results: 1.The experimental results of orthogonal design showed that the best conditions were feeding with high protein and calorie diet for 8 w and 12 mg/kg loperamide for 10 d.The rat model of GHRS is successfully established.2.Results of the model validation experiment showed that compared with the control group,the rats in the GHRS showed a decrease in diet,an increase in abdominal circumference and body weight ratio,a deep reddish tongue phase,yellow urine,constipation,decreased bowel sounds times,elevated body temperature,willing to stay in cold circumstance,and significant symptoms of inflammatory response,Niuguang Jiedu tablets can alleviate these symptoms.It shows that the model of GHRS is established successfully.3.Realgar can reduce the ratio of abdominal circumference to body weight in GHRS group.And the urine became pale yellow,the stool was soft and the score was decreased,the frequency of bowel sounds increased,and the residence time in low temperature area decreased.The results of arsenic content determination showed that the content of arsenic in hippocampus increased in 1.8g/kg realgar group,GHRS+0.3g/kg realgar group and GHRS+1.8g/kg realgar group.The results of transmission electron microscope showed that the hippocampal neurons of GHRS group and realgar group had different degrees of nuclear morphological abnormalities.And mitochondrial ultrastructure,swelling,and vacuolation were observed.In realgar group,the myelin sheath structure was damaged and the synaptic structure was abnormal.GHRS+0.3g/kg realgar group had less injury.The expression of Iba-1 was significantly increased in GHRS group(P<0.05)and it was significantly decreased in GHRS+0.3g/kg realgar group(P<0.05).The positive expression of NF-?B p65 was increased in nucleus in GHRS group(P<0.05).And it was decreased in GHRS+0.3g/kg realgar group and GHRS+1.8g/kg realgar group.The expression of IL-6 was decreased significantly in GHRS+0.3g/kg realgar group(P<0.05).The expression of claudin-5 and occludin were decreased significantly in GHRS group(P<0.05).The expression of occludin was decreased significantly in realgar group(P<0.05).The expression of MMP-9 was increased significantly in GHRS and realgar group(P<0.05)and it was decreased significantly in GHRS+0.3g/kg realgar group and GHRS+1.8g/kg realgar group(P<0.05).The expression of TNF-? in serum increased significantly in GHRS group(P<0.05).4.The results of metabonomics showed that realgar could change the levels of 23 metabolites in hippocampus of rats with GHRS,the levels of 7 metabolites were increased(P<0.05),such as choline,and the levels of 16 metabolites were decreased(P<0.05),such as meso-tartaric acid,8 of them can be used as potential metabolic markers.Glutathione and histidine can be used as sensitive metabolic markers to judge the effect of realgar on hippocampus of rats with GHRS.These altered metabolites may disturb some metabolic pathways,including glutathione metabolism,histidine metabolism,glutamic acid metabolism,glycerin phospholipid metabolism,purine metabolism,and energy metabolism.The results of fecal microbiome showed that compared with GHRS group,at the species level,the abundances of Papillibacter,Lachnospira,Bacteroides uniformis,and Sellimonas were significantly decreased(P<0.05),and the abundance of Rikenellaceae RC9 gut group was significantly increased(P<0.05).Administration of realgar antagonized an increase in fecal microbial abundance of Papillibacter,Sellimonas,and Lachnospira in rats with GHRS.The levels of choline and histidine in the hippocampus were increased.The levels of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate,ureidoglycolate,glutathione,and ascorbate were decreased.Realgar was able to antagonize damage to the cell membrane and myelin sheath structures,oxidative damage and inflammatory reaction of hippocampal neurons caused by GHRS.Glutathione and histidine in the hippocampus and fecal microorganisms,such as Papillibacter,Lachnospira,and Sellimonas,were the sensitive targets of realgar in GHRS rats.5.When realgar enters normal rats,it can change the levels of 16 metabolites.Compared with the control group,the levels of 12 metabolites were increased(P<0.05),such as Acetyl-Co A,and the levels of 4 metabolites were decreased(P<0.05),such as creatine,9 of them can be used as potential metabolic markers.5-Hydroxy-2-furoic acid,3-hydroxypropanoyl-Co A,and creatine can be used as early metabolic markers to assess the effects of realgar on the hippocampus of normal rats.These metabolites are mainly involved in purine metabolism,arginine and proline metabolism,?-alanine metabolism,and the tricarboxylic acid cycle pathway.The results of fecal microbiome showed that compared with the control group,at the family level the abundance of Alcaligenaceae was significantly increased(P <0.05).At the genus level,the abundance of Parasutterella was significantly increased(P<0.05),and the abundance of a gram-negative bacterium c TPY?13 was significantly decreased(P <0.05).Administration of realgar to normal rats increased the abundance of Parasutterella in the feces and the expression of IL-6 in the serum.Realgar induced inflammatory reactions,reduced the levels of PE,ureidoglycolate,and creatine in the hippocampus,and increased the levels of Acetyl-Co A and3-hydroxypropanoyl-co A,resulting in damage to the cell membrane and myelin sheath structures of neurons,abnormal energy metabolism,and inflammatory reactions.3-Hydroxypropanoyl-Co A is the target of arsenic in the host and in Parasutterella.Conclusion: 1.The rat model of GHRS was successfully established.The best conditions were feeding with high protein and calorie diet for 8 w and 12 mg/kg loperamide for 10 d.2.Realgar has antagonistic effect on the changes of macroscopical characteristics caused by GHRS in rats,which shows therapeutic effect.3.Realgar has neuroprotective effect on hippocampus of rats with GHRS.Glutathione and histidine are sensitive metabolic markers of realgar on hippocampus of rats with GHRS.Realgar has neurotoxic effect on hippocampus of normal rats.5-Hydroxy-2-furoic acid,3-hydroxypropanoyl-Co A,and creatine are sensitive metabolic markers of realgar on hippocampus of normal rats.4.Realgar has therapeutic effect on fecal flora imbalance caused by GHRS.Papillibacter,Lachnospira,and Sellimonas,were the sensitive targets of realgar in GHRS rats.Realgar can cause imbalance of fecal microflora in normal rats.Parasutterella were the sensitive targets of realgar in normal rats.5.Realgar regulates the balance of blood-brain barrier and the level of metabolites in hippocampus of rats by regulating the composition of fecal flora and mediating the MGB axis.Realgar has both protective and toxic effects on the central nervous system of GHRS rats and normal rats. |
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