| Objective: Realgar contains arsenic and is one of the 28 toxic traditional Chinese medicines specified in the "Administrative Measures on Toxic Drugs for Medical Use of the People’s Republic of China".Clinically,chronic arsenic poisoning caused by the abuse of Realgar-containing Niuhuang Jiedu Tablets has occurred from time to time,manifested as multi-system damage throughout the body.The central nervous system is one of the target organs of its toxic effect.Safety has been widely questioned.Therefore,the issue of the removal/retention(toxicity/effect)of realgar in Niuhuang Jiedu tablets has become the focus and hot spot of people’s attention.Niuhuang Jiedu Tablet is a representative medicine for the treatment of gastrointestinal heat syndrome.Gastrointestinal heat syndrome is related to inflammatory damage caused by imbalance of immune factor secretion and dry stool caused by imbalance of intestinal flora.The intestinal flora can regulate the activity of the central nervous system of the brain through the gut-brain axis.When the intestinal flora is imbalanced,inflammatory factors such as NF-κB and IL-6 in the blood attack the blood-brain barrier,causing microglia and MMP-9 to be activated,Iba-1 is highly expressed,and MMP-9 is further used as hydrolysis Protease acts on the tight junction of the blood-brain barrier,resulting in a decrease in the expression of Occludin and Claudin-5,an increase in the permeability of the blood-brain barrier,and the destruction of the structure of the bloodbrain barrier.So,does realgar have the same effect on the cerebral blood-brain barrier under pathological and physiological conditions? There is no report so far,so it is necessary to re-evaluate the toxicology of realgar to provide theoretical basis and experimental data for the re-understanding of the toxicology of realgar in the classic prescription Niuhuang Jiedu Tablets.Methods: SPF female SD rats were randomly divided into control group(CON),gastrointestinal hyperthermia model group(TREAT),gastrointestinal hyperthermia model +1.8 g/kg realgar group(TRH)and 1.8 g/kg realgar group(REA)according to body weight,with 8 rats in each group.1.Intestinal sound measurement: the number of intestinal sounds in 5 min was recorded.2.Determination of defecation: The feces of rats were collected and scored using the Bristol feces classification.3.Measurement of abdominal girth/body weight ratio: The abdominal girth of rats was recorded,the weight of rats was weighed,and the abdominal girth/body weight ratio of rats was calculated4.Determination of tongue color: photos of the tongue of rats were taken,and Photoshop image processing software was used to cut the photos of the tongue of rats into appropriate pictures of the tongue.Nine coordinate points were set to record the values of red,green and blue pixels at the points,and the mean values of pixels at each coordinate point were calculated.5.Urine color determination: urine of rats was collected and urine photos were taken.Photoshop image processing software was used to record the blue pixel value at the midpoint of each hole.6.Measurement of ear swelling rate: After the right ear was treated with xylene for 30 min,the left and right ear pieces of rats were knocked down with a hole punch,weighed and calculated the swelling rate.7.Determination of anal temperature: The rats were fixed in the cage,and after stabilization,the anal temperature was measured with a special rat anal temperature detector and recorded.8.Measurement of the activity rate of hot and cold trend: After the rats were trained to get familiar with the environment,the rats were placed in the hot and cold plate system at room temperature(25℃)for 5 min,and the specific residence time of the rats in the cold plate and the hot plate was recorded respectively.9.Western Blot was used to detect the protein expression levels of NF-κB,IBA-1,IL-6,MMP-9,Occludin and Clauid N-5 in each group.10.Statistical analysis: The experimental data are expressed as mean±standard deviation((?)±SD),graph Pad Prism7.0 software is used for drawing,and one-way analysis of variance(One Way ANOVA),multi-factor analysis of variance(Two Way ANOVA)and Dunnett’s method are used for multiple groups The statistical analysis of the significance of the difference showed that the difference was statistically significant when P<0.05.Results: 1.Compared with the CON group,the Treat group had dry and hard feces,yellow urine,dark tongue color,increased abdominal circumference,increased anal temperature,reduced number of intestinal sounds,hypothermia,and increased ear swelling rate.After realgar intervention,compared with the Treat group,the stool quality of the rats in the TRH group became soft,the tongue phase color recovered,the urine color became lighter,the abdominal circumference decreased,the anal temperature decreased,the number of intestinal sounds increased,the hypothermia condition improved,and the ear swelling rate decreased.Compared with the CON group,there were no significant changes in feces,bowel sound frequency and anal temperature in the REA group.The urine color became darker,the tongue color became lighter,the abdominal circumference decreased,and the ear swelling rate increased.The results showed that realgar had an antagonistic effect on the macroscopic changes caused by gastrointestinal heat accumulation syndrome in rats,but had a weak effect on the macroscopic changes of normal rats.2.Compared with the CON group,the expression levels of p65,IL-6,Iba-1and MMP-9 protein in the cerebral cortex of rats in the TREAT group and the REA group increased(P<0.05),and the protein expression levels of Occludin and Clauidn-5 After the intervention of realgar,compared with the TREAT group,the expression levels of p65,IL-6,Iba-1 and MMP-9 protein in the cerebral cortex of the TRH group decreased(P<0.05),Occludin,Clauidn-5 protein expression level increased(P<0.05).The results indicated that realgar had a certain protective effect on the central nervous system of rats with gastrointestinal heat syndrome,and had a certain toxic effect on the central nervous system of normal rats.Conclusion: 1.Gastrointestinal fever syndrome can cause abnormal macroscopic indicators,inflammation,and increased permeability of the blood-brain barrier in rats,suggesting that gastrointestinal fever has a certain harmful effect on the central nervous system of normal rats.2.Realgar has an antagonistic effect on the abnormal macroscopic indicators,inflammatory response,and increased permeability of the blood-brain barrier in rats caused by gastrointestinal heat syndrome,suggesting that realgar has certain protective effects on the central nervous system of rats with gastrointestinal heat syndrome.3.Realgar can increase the permeability of the blood-brain barrier in normal rats and increase the inflammatory response,suggesting that realgar has a certain toxic effect on the central nervous system of normal rats. |
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