| Rhei Radix et Rhizoma is a commonly used traditional Chinese medicine,and it is a dry rhizome and root of the family Rheum tanguticum Maxim.ex Balf.,Rheum offcinale Baill..and Rheum palmatum L.with diarrhea.Under attack the plot,heat and purging fire,cooling blood detoxification,by phlegm pass through and dampness jaundice and other effects,clinical application of real heat stagnation constipation,blood spit diarrhea,red eyes,pharyngeal swollen hemorrhoids,intestinal fistula Abdominal pain,blood stasis,postpartum obstruction,bruises,hot and humid diarrhea,jaundice,urine red,shower,edema,external burns[1].The major components of rhubarb are steroids,including emodin,rhein,chrysophanol,and emodin,in addition to benzophenone glycosides,tannins,volatile oils,polysaccharides,fatty acids,and proteins[2]Rhubarb is bitterly cold,and the power of diarrhea is rapid.If it is used improperly,it may cause adverse reactions[3].Previous studies on the toxicity of rhubarb and its main component,steroids,have been reported.The conclusions are somewhat different.Many believe that fleas may cause hepatotoxicity at a certain dose.However,there have been few studies on the hepato-renal toxicity of rhubarb and wolfberry in the past.Based on the toxicological studies of rhubarb and its total glycosides,the study focused on the effect of rhubarb on the expression of liver and kidney transporter and explored its toxicity mechanism.According to previous studies,the toxic dose of rhubarb extract ranged from 10 to 12 g/kg,and the toxic dose of total rhubarb was from 135 mg/kg to 4500 mg/kg.In the first part of this study,we will design an experimental dose with reference to this toxic dose range,and repeat the liver and kidney toxicity studies of rhubarb extract and total carp.Liver and kidney are the body's metabolic and excretory organs,respectively.They play an important role in the metabolism and transport of endogenous substances and exogenous substances including drugs.The second part of this study will mainly observe the effect of rhubarb and its total glycosides on the expression of liver-kidney-related transporters to explore the mechanism of liver and kidney toxicity.Objective:To observe the liver and kidney toxicity of rhubarb extract and total saponins at different doses and to understand the toxic dose range;To investigate the mechanism of liver and kidney toxicity of rhubarb extract and total glycosides through analysis of liver and kidney related transporter mRNA.Methods:Eighty Sprague-Dawley Sprague-Dawley rats were male and female.According to body weight,they were randomly divided into control group A and rhubarb extract different dose groups(2,8 and 16 g crude drug/kg,which were equivalent to 8,32,and 64 times of clinical dose,respectively);control group B and rhubarb were given different doses.Group(0.5,2.5,5 g powder/kg).Each group had 10 animals,5 males and 5 females.Rhubarb extract different dose groups were administered by gavage,once daily,continuous administration for 1 month.Control group A and rhubarb different dose groups were tested in parallel and an equal volume of purified water was given.Rhubarb total glycosides were intragastrically administered in different dosage groups for 10 days once daily for 10 consecutive days.Control group B was tested in parallel with total rhubarb,and an equal volume of purified water was given.After the end of the last administration,16h urine was collected using a metabolic cage to measure the amount of urine.The urinary analyzer was used to determine the urine qualitative indicators(urine protein,urine glucose,urobilinogen,red blood cells,white blood cells,etc.);urine was measured by ELISA.The content of NGL(Neutrophilated gelatinase-associated lipocalin)and KIM-1(Kidney injure molecule-1).The animals were anesthetized and blood was collected from the abdominal aorta.The serum was separated and analyzed for serum biochemical and hematological parameters such as ALT,AST,BUN,and CRE.The liver and kidney were removed,and part of the liver and kidney tissues were fixed with formaldehyde,extracted,dehydrated,embedded in paraffin,sectioned,stained with hematoxylin-eosin and pathologically examined under a microscope.The other part of the kidney tissue was divided into RNase-free test tubes,frozen in liquid nitrogen,and stored at 80? for subsequent RT-PCR assay.The renal organic anion transporters(OAT1,OAT3)and kidneys were analyzed.Gelatin Gelase-associated Lipocalin(NGAL),Clusterin mRNA Expression Levels.Another group of animals was divided into control group and total rhubarb 5 g/kg dose group for the detection of amino-hippuric acid(PAH)clearance rate test.Rats were given intragastric administration of total rhubarb 5 g/kg for 10 days and then anesthetized with intraperitoneal injection of 10%chloral hydrate(350 mg/kg rats).Under aseptic conditions,the common carotid vein was cannulated and thefemoral vein was cannulated.Rats with successful operation were placed in single cages in IVC cages and recovered after surgery.After fasting for 12-16 h(freedrinking water),30 mg/kg PAH solution at a concentration of 6 g/L was injected from the jugular vein,and the IV infusion concentration was 3 g/L PAH solution 6mg/h/100 g for 45 min.Then,timing was started and blood samples were collected at 1,5,10,15,20,25,30,40,50,and 60 min in the conscious state of the rats.After each time point of blood collection,the same volume of physiological saline wasintravenously input.Rat urine was collected at 20,40,60 min and the urine volume was measured at each time period.The PAH content in blood and urine was analyzed by HPLC to calculate the PAH clearance rate.Results:1.Rhubarb extract and total rhubarb doses can cause mild kidney damage,but no significant liver and nephrotoxicity at medium and low doses.Rats were given intragastric administration of rhubarb extract high-dose group(16 g crude drug/kg,equivalent to 64 times the clinical dose).After administration,soft stools,unclean hair,dullness,and deepening of urine color were observed.Creatinine(CRE)levels were higher than those of the control group(p<0.001);kidney weight of the male rats was significantly lower(p<0.05).There were no obvious toxicological changes in other biochemical indicators and low-to-medium dose biochemical indicators in the high-dose group.Pathological examination showed that in the high-dose group(16 g crude drug/kg),there was a slight focal inflammatory cell infiltration in the liver,but there was no significant difference compared with the control group.Therefore,toxicological significance was not considered.In the high-dose group,the kidneys of male and female rats showed mild or moderate tubular cell swelling and degeneration,and a small amount of inflammatory cells infiltrated.The kidney lesions of male rats were slightly heavier than female rats,and there were 2 tabular inflammations in male rats.Infiltration of cells with fibroblast proliferation.No liver and kidney pathological lesions were found in the middle and low dose groups.After the administration of rhubarb total wolfberry in the high-dose group(5 g/kg),the body weight of the rats decreased significantly,which may be related to the decrease of feed intake.After the administration,the stool is soft,the coat is not clean,and the hair is not shiny.On the 4th day after administration,the rats in the high-dose group showed delayed movement and apathy,and the male rats died on the 7th day.Therefore,on the 10th day after the administration,the animals in each dose group were sacrificed for examination and analysis.Blood creatinine increased in the high-dose group(5 g/kg)(p<0.01),an increase of 162.5%compared with the control group.Kidney weight and kidney index were significantly lower in the high-dose group than in the control group(p<0.001).Pathological findings showed.In the high-dose group,a small amount of hepatocyte eosinophilic enhancement and nuclear staining were observed in the liver,but this phenomenon was also seen in the control group,so the toxicological significance was not obvious.In the high-dose group,slight swelling of the renal tubules and infiltration of inflammatory cells were observed,however,no obvious pathological changes were observed in the liver and kidney of the low-and middle-dose groups.2.Effects of Rhubarb Extract and Total Rhubarb on Urine NGAL and KIM-1 Levels in RatsOne month after intragastric administration of Rhubarb extract to rats,the urinary NGAL levels of the male animals in the high-dose group and the low-dose group increased,and there was a significant difference between the high-dose group and the normal group(p<0.05).Liquid NGAL showed no significant effect.The combined data of males and females showed that the levels of NGAL in the urine of the low-dose group and the high-dose group were respectively increased by 22.78%and 51.53%compared with the normal group.Among them,the two dose levels have a stronger effect on the elevation of NGAL in male urine than females.The level of KIM-1 in the urine of male and female rats of high-dose rhubarb group was significantly lower(p<0.01 compared with the normal group),but it was considered to have no toxicological significance.After rats were given intragastric administration of rhubarb for 10 days,the levels of urinary NGAL in male and female animals in each dose group were significantly increased,which may be related to the greater amount of total indole in each dose group.There was no toxicological change in KIM-1 levels.3.Effects of rhubarb extract and total rhubarb on expression of Clusterin,OAT1 and OAT2 mRNA in kidneyRhubarb extract was intragastric administration for one month.The expression of Clusterin mRNA in kidney tissue of high-dose group was significantly lower than that of normal group(p<0.01),and the male and female animals were decreased by 33%and 19%respectively compared with the normal group.There was no significant change in the expression of Clusterin mRNA in the kidney tissue of the low-dose group.The expression of OAT1 mRNA and OAT3 mRNA in the kidney-tissue of the male rhesus low-dose group was increased by 47%and 62%,respectively,compared with the normal group.OAT3 mRNA was up-regulated by 38%in the high-dose group compared with the normal group.After administration of rhubarb in each dose group,there was no significant change in OAT1 mRNA and OAT3 mRNA expression in the kidney tissue of female animals.Rhubarb was administered intragastrically for 10 days.The expression of Clusterin mRNA in kidneys of female rats in the medium and high dose groups was decreased by 290%and 26%compared with the normal group.The mRNA levels of OAT1 in the kidneys of female and male animals in the medium and high dose groups were reduced by 21%,42%,43%,and 70%,respectively,compared with the control group.The expression of OAT3 mRNA in the kidneys of high-dose female animals was reduced by 44%compared with the normal group.4.Effect of total rhubarb on the clearance rate of amino-hippuric acidThe PAH clearance experiment was performed after 10 days of intragastric administration of total rhubarb to 5 g/kg.The results showed that the concentrations of PAH in plasma of the male and female animals in the administration group at each time point were lower than those in the control group.The content of PAH in urine of male animals was lower in the first 20 min after administration of PAH than in the control group.The content of PAH in female urine was higher than that in the control group at 20-40 min and 40-60 min.The PAH clearance rate of 20-40 min and 40-60 min in Rhubarb group was lower than that in the control group.The results suggest that rhubarb 5 g/kg intragastric administration for 10 days will inhibit the rat kidney anion transporter,which will reduce the PAH clearance rate and may causeaccumulation of anionic substances in the kidney.Conclusion:There was no obvious hepatotoxicity and renal toxicity in the low and middle dose groups of rhubarb and rhubarb.Rhubarb extract high-dose group(16 g/kg)and high-dose rhubarb group(5 g/kg)can cause mild renal damage.Both had no obvious toxic effects on the liver under the experimental conditions.The traditional indicators of renal function tests BUN and CRE failed to reflect the kidney injury caused by high doses of rhubarb and rhubarb,and increased urine NGAL analysis may improve the objectivity of its detection.Clusterin is a newly discovered anti-apoptotic factor in recent years,which protects renal cells.Rhubarb and Rhubarb can down-regulate the expression of Clusterin mRNA in renal tissue at high doses,which may inhibit the body'santi-apoptotic capacity and reduce the protective effect on the kidneys,thus increasing the risk of kidney damage.High doses of Rhubarb can reduce OAT1 and OAT3 mRNA expression in kidney tissue and reduce the clearance rate of PAH,suggesting that the kidney toxicity caused by Rhubarb dose may be too high and it may inhibit the function of organic anion transporter,leading to harmful substances in the kidney.Medium accumulation related. |
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