| Objectives:Inonotus obliquus(Fr.)is a rare and valuable medicinal fungus,which mainly parasitizes under the bark of broad-leaved trees such as birch,silver birch,elm,alder,etc.or on the dry trunks of the above trees after felling.At present,domestic and foreign scholars have conducted preliminary studies on the pharmacological effects of Inonotus obliquus in antitumor,immunomodulation,hypoglycemia,hypolipidemia,antioxidant,anti-fatigue and other aspects,but the studies on the biosafety,toxicity and toxicological mechanisms of Inonotus obliquus still remain at the general toxicity evaluation level,lacking in the exploration of toxicity mechanisms,and the small molecule metabolites and metabolic pathways closely related to the toxicity of Inonotus obliquus.So it is necessary to carry out further study on the mechanism of toxicity.By using traditional toxicological methods combined with metabonomics,the toxicity characteristics of SD rats were characterized by repeated administration of Inonotus obliquus.the target organs of toxicity were explored,and the mechanism of toxicity of Inonotus obliquus was investigated by applying traditional toxicological methods in combination with metabolomics techniques and other systemic toxicological methods.In the present study,we investigated the toxicological effects of repeated administration of Inonotus obliquus using traditional toxicological methods to reveal the potential target organs of Inonotus obliquus,including the effects of Inonotus obliquus on body weight,food intake and organ coefficients,clinical hematology,serum biochemistry and urine parameters,and organ histopathological changes in SD rats.On the other hand,modern metabolomics technology was used to explore the mechanism of toxicity of Inonotus obliquus starting from the analysis of small molecule metabolites;Finally,the results of traditional toxicology studies and metabolomics studies were organically integrated and analyzed to obtain information on the toxicological characteristics and toxicity mechanism of Inonotus obliquus.Through the implementation of this study,firstly,toxicological information on the toxicity characteristics and toxicity mechanism of the repeated administration of Inonotus obliquus will be obtained;secondly,it will provide a scientific basis for the establishment of safety evaluation criteria of Inonotus obliquus and accumulate practical experience for exploring the toxicological research methods of Inonotus obliquus.Methods:The toxicity test LD50 of alcohol extract of Inonotus obliquus was 5865 mg/kg weight,Human clinical dose:1000 mg/d(13mg/kg body weight)and the Technical Guidelines for Toxicity Tests for Repeated Drug Administration issued by the State Food and Drug Administration in May 2014.The alcohol extract of Inonotus obliquus was converted into three dose groups according to the volume of administration in rats(1ml/100g body weight)and the frequency of administration per day(1/day),500,1000,2000 mg/kg/d(about 38.5,77,154 times the daily dose),A solvent control group(pure water of the same volume).Each group of 20 SD rats,10male and female,once a day,7 days a week,13 consecutive weeks.The appearance,behavior,body weight and food intake of rats were tested during the experiment.Hematology,blood biochemistry and urine routine were tested at the end of 13 weeks.Results:(1)Effect of alcohol extract of Inonotus obliquus on clinical diagnosis of SD ratsBlack stool appeared in the second week after administration of high dose SD rats of Inonotus obliquus alcohol extract.Because the alcohol extract of Inonotus obliquus is brown and black when prepared,the black stool of the animal is related to the alcohol extract of Inonotus obliquus.The feces of the solution showed black after digestion in animal gastrointestinal tract.Compared with the control,the intake of alcohol extracts from Inonotus obliquus SD male rats decreased in varying degrees.The difference between 2?4,6,7?9 and 13 weeks after administration was significant(P<0.05)or very significant(P<0.01).High-dose male SD rats not only had lower body weight than non-administered control rats,but also had lower body weight than low-dose and medium-dose rats.The difference between 2,4 and 10?13 weeks after administration was significant(P<0.05).All the SD rats were treated with the alcohol extract of Inonotus obliquus,and the ratio of spleen to viscera was significantly(P<0.05)higher than that of control rats.The alcohol extracts of Inonotus obliquus were given to SD rats,and the ratio of spleen body mass index was significantly higher(P<0.05)than control rats.Compared with the control rats,Inonotus obliquus alcohol extract was given to male SD rats whose organ body mass index increase,except adrenal gland.was significantly(P<0.05)or extremely significantly(P<0.01)higher than that in control rats,and the ratio of testis to epididymal organs in high dose male rats was significantly(P<0.05)higher than that in low dose and medium dose rats.The organ body mass index of testis and epididymal in SD rats after administration was significantly(P<0.05)or extremely significantly(P<0.01)higher than that in control rats,and the organ body mass index of testis in high dose male rats was significantly(P<0.05)higher than that in low dose and medium dose rats.When Inonotus obliquus alcohol extract is given to female SD rats,its uterus body mass index is significantly(P<0.05)or extremely significantly(P<0.01)higher than that of the control group and the low-dose group,and the high-dose group is significantly(P<0.05)higher than that of the low-dose group.Inonotus obliquus alcohol extracts were given to SD rats,and the ratio of organ brain index was higher than that of control rats in different degrees,and the ratio of organ brain index in liver was significantly higher(P<0.05)than that of control rats.The ratio of organ brain index in testis in male SD rats was significantly higher in high dose rats(P<0.05)than in medium dose rats.The ratio of organ brain index in uterus in female SD rats was significantly higher(P<0.05)or extremely significant(P<0.01)in medium and high dose than that in control and low dose rats.(2)Effect of Inonotus obliquus alcohol extract on physical and chemical indexes of SD ratsThere was no significant difference between the other groups except that the high dose of Inonotus obliquus alcohol extract was given to male SD rats,and the urine WBC and p H were significantly reduced compared with the medium dose(P<0.05),while the high dose of Inonotus obliquus alcohol extract was given to female SD rats,and the urine white blood cells were significantly reduced compared with the control rats(P<0.05).When the alcohol extract of Inonotus obliquus was given to male SD rats,the number of RBC and WBC in blood increased with the increase of dosage,while the percentage of MONO decreased with the increase of dosage.The alcohol extract of Inonotus obliquus was given to female SD rats,the MCV,MCH and RET values in blood increased significantly with the increase of dosage.NEUT percentage which is Inonotus obliquus alcohol extract high-dose group was significantly higher than the control group and medium-dose group.LYMPH percentage which Inonotus obliquus alcohol extract high-dose group was lower than the control group,low-dose group and medium-dose group.MONO percentage decreased significantly with the increase of dosage.Inonotus obliquus alcohol extract is given to SD rats,there is no statistical difference in blood coagulation factors.Inonotus obliquus alcohol extract is given to SD rats,its serum TG and CHOL contents are lower than those of control rats,and its value changes have a certain negative correlation with the dosage of Inonotus obliquus alcohol extract,but there is no statistical difference.The alcohol extracts of Inonotus obliquus were given to rats respectively,and their blood TBIL,TP,ALB values were lower than those of control rats.except that TBIL values of high dose male SD rats,middle dose and high dose rats were significantly lower than those of control rats(P<0.05),there was no significant difference between the other groups(P>0.05).Inonotus obliquus alcohol extract is given to SD rats,the serum CK value of high dose rats is significantly lower(P<0.05)than that of other groups of rats,while AST value is significantly lower(P<0.05)than that of control rats,and the rest have no statistical difference(P>0.05).Inonotus obliquus alcohol extract is given to SD rats,the serum Crea content is reduced to different degrees compared with the control group,among which the low-dose male SD rats are significantly reduced(P<0.05),and the high-dose female SD rats are particularly significantly reduced(P<0.01).Compared with the control rats,the Urea content in serum of male SD rats treated with alcohol extract of Inonotus obliquus increased to different degrees,and the high dose rats were significantly(P<0.05)or extremely significantly(P<0.01)higher than the control,low dose and medium dose rats.Inonotus obliquus alcohol extract is given to the male SD rats at low dose,its Glu content was significantly lower(P<0.05)than that of control rats.And at high dose in SD rats,its Glu content was not only extremely significantly lower(P<0.01)than that of control rats but also significantly lower(P<0.05)than that of low-dose and medium-dose rats,while the rest showed no significant difference(P>0.05).(3)Effect of Inonotus obliquus alcohol extract on organ histopathological changes in SD ratsCompared with the control group,the medium and high doses of Inonotus obliquus alcohol extract were given to SD rats,histopathological changes such as inflammatory cell infiltration,bile canaliculus epithelial swelling,liver cell atrophy and so on were observed in different degrees in the liver.When high dose is given to rats,histopathological changes such as inflammatory cell infiltration,renal tubular epithelial swelling,interstitial hyperplasia,etc.can also be seen in the kidneys.Inonotus obliquus alcohol extract was given to male rats at medium and high doses,and its prostate showed inflammatory cell infiltration in different degrees.Uterine glands and endometrium of female rats are hyperplastic,etc.(4)Effects of Inonotus obliquus alcohol extract on sperm morphology and motility in ICR miceThe alcohol extract of Inonotus obliquus was given to ICR mice,the sperm deformity rate of mice in each administration group and control group was significantly lower(P<0.01)than that in the positive drug group,and there was no statistical difference between the other groups(P>0.05).Through computer-assisted sperm analyzer detection,compared with the control mice,there was no statistical difference in all tested indexes of sperm motility ability of Inonotus obliquus alcohol extract to ICR mice(P>0.05),and there was no difference change between each administration group(P>0.05).(5)Effect of Inonotus obliquus alcohol extract on chromosomal mutation of ICR miceThe micronucleus test showed that the micronucleus rate of chromosome in the positive control group was significantly higher than that in the alcohol extract of Inonotus obliquus and the negative control group(P<0.01),while there was no statistical difference in the rest(P>0.05).(6)Changes in metabolic profile of potential biomarkers after Inonotus obliquus alcohol extract is given to SD ratsAfter the application of the alcohol extract of Inonotus obliquus in SD rats,according to the results of S-plot and VIP-plot,the differential variables with VIP>1 were selected,and then the t-test was used to analyze the obtained differential variables between groups,only the differential variables with P<0.05 were considered as potential biomarkers.Among them,8 were up-regulated and 31 were down-regulated.through Meta PA identification,significant enrichment pathway analysis showed that these targets were involved in the regulation of 10 metabolic pathways including arginine biosynthesis,glyceride metabolism,phosphatidylinositol signaling system,cysteine and methionine metabolism,glycerophospholipid metabolism,arginine and proline metabolism,pyrimidine metabolism,primary bile acid biosynthesis,aminoacyl-t RNA biosynthesis,steroid hormone biosynthesis,etc.It is found that 38 differential metabolites exist in potential intervention targets in serum,Among them,15 are up-regulated and 23 are down-regulated.Through Meta PA identification,significant enrichment pathway analysis,such targets are involved in the regulation of 10 metabolic pathways such as glycerophospholipid metabolism,arachidonic acid metabolism,linoleic acid metabolism,?-linolenic acid metabolism,glycosyl phosphateidylinositol(GPI)-anchored biosynthesis,glyceryl lipid metabolism,pentose and gluconic acid mutual conversion,sphingolipid metabolism,phosphatidylinositol signal system,primary bile acid biosynthesis,etc.It is found that 44 potential differential metabolic product intervention targets in the liver,20 are up-regulated and 24 are down-regulated.Through Meta PA identification,significant enrichment pathway analysis,such targets are involved in the regulation of 10 metabolic pathways including glycerophospholipid metabolism,linoleic acid metabolism,?-linolenic acid metabolism,glycosyl phosphateidylinositol(GPI)-anchored biosynthesis,glyceride metabolism,steroid hormone biosynthesis,sphingolipid metabolism,phosphatidylinositol signaling system,glutathione metabolism,arachidonic acid metabolism,etc.It is found that 28potential differential metabolic product intervention targets in the kidney,11 are up-regulated and17 are down-regulated.Through Meta PA identification,significant enrichment pathway analysis,such targets are involved in the regulation of 8 metabolic pathways including glycerophospholipid metabolism,linoleic acid metabolism,primary bile acid biosynthesis,taurine and taurine metabolism,?-linolenic acid metabolism,glycosylphosphatidylinositol(GPI)-anchored biosynthesis,sphingolipid metabolism,arachidonic acid metabolism,etc.Conclusions:1.Alcohol extract of Inonotus obliquus was given to SD rats,which changed the related pathways of lipid metabolism and liver microcirculation metabolism.2.Inonotus obliquus alcohol extract was given to ICR mice and no reproductive and genotoxicity were found.3.Subchronic toxicity test combined with metabonomics study showed that liver and kidney were the main target organs of alcohol extract of Inonotus obliquus. |
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