| Research objectTo explore the possible toxic site and its mechanism of Radix Sophorae Tonkinensis(RST),and to explore whether the compatibility with Rhizoma Belamcandae(RB)and Rhizoma Cimicifugae(RC)has the effect of reducing toxicity.Research BackgroudRST is a common clinical drug used to clear heat and promote pharynx,but in recent years,it has been found to have obvious liver toxicity and cardiovascular toxicity."Pei Wu" means that the side effects of a certain drug in the combined drug group can be inhibited,alleviated or eliminated by other drugs,in order to achieve the purpose of restraining bias and achieving the purpose of safe use of drugs.This study intends to improve the safety of clinical drug use of RST by compatibility study.The mixed decoction was first recorded in "Fuji recipe"(Volume 60,named"Shandou Root recipe")of the Ming Dynasty.Both of RST and RB are important drugs to clear heat and promote pharynx.RB can lead to Yangming Qingqi uplink,which has the auxiliary effect of dialystics and divergence.According to the traditional prescription,the combination of three drugs can be used to reduce the toxicity.The problems to be solved in this study are:whether the mixed decoction ngma has the effect of reducing poison?What is the toxic components of RST?What might be the mechanism of hepatotoxicity caused by the RST?Therefore,the acute toxicity test of mice,the acute toxicity test of zebrafish and the cytotoxicity test were used to evaluate the antitoxic effect of the mixed decoction.The acute toxicity test of zebrafish and cytotoxicity test were used to screen the toxic components of RST.The mechanism of damage caused by the toxic site was simply explored by LDH release test and apoptosis test.The blood biochemistry of mice was determined after repeated administration for 7 days.The expression of CYP450 mRNA was detected by QRT-PCR method.Western Blot assay was used to detect the expression of CYP450 protein.All of this can be used to explore the possible mechanism of hepatotoxicity of the root.Research content1.Study on the antitoxic effect of mixed decoction1)100 ICR mice of SPF grade were randomly divided into 10 groups(Control,single decoction group,combined decoction group,negative group),Each group has 5 female and 5 male mice.The dosage of mice was 12.3、15.3、19.2、24.0 g kg-1,respectively.We closely observed the toxic reaction of animals after the first administration,and observed it once a day for the next 14 days.We observed whether there has some toxic reaction,toxic symptoms and its degree,the time of toxicity appearing and disappearing,and recorded the number and time of death of animals.And the LD50 was calculated in the end.2)Acute toxicity of zebrafish at embryonic stage:the final concentrations were 0.156、0.312、0.625、1.25、2.5、5 g·L-1,respectively.The final concentrations in the mixed decoction were 0.0625、0.125、0.25、0.5、1 g-L-1,,respectively.The death and deformity of zebrafish embryos were observed at 24 h and 48 h after administration,and the death number was recorded and LC50 was calculated.Acute toxicity of zebrafish in yolk sac stage:the final concentration of RST was 0.156、0.312、0.625、1.25、2.5、5 g·L-1,and the final concentration in the mixed decoction were 0.039、0.078、0.156、0.312、0.625、1.25、2.5 g·L-1,respectively.The death and abnormality of zebrafish embryos were observed at 24 h and 48 h after administration,the mortality and abnormality of zebrafish embryos were recorded and LC50 was calculated.3)HepG2,H9c2 cells were cultured in vitro.The concentration were 1.25、2.5、5、10、20、40 g·L-1.MTT was used to detect cell activity,the toxicity was measured by calculating IC50.2.Study on the effects of different extracts of RST1)Acute toxicity of zebrafish at embryonic stage:the final concentration of non-alkaloid was 0.125、0.25、0.5、1、2 g·L-1,and the final concentration of alkaloid was 1、2、4、8、16 g·L-1,respectively.The death and deformity of zebrafish embryos were observed at 24 h and 48 h after administration,the death number was recorded and LCso was calculated.In the acute toxicity of zebrafish at yolk sac stage,the final concentrations of nonalkaloid and alkaloid were 0.312、0.625、1.25、2.5 g·L-1,respectively.The death and deformity of zebrafish embryos were observed at 24 h and 48 h after administration,and the death number was recorded and IC50 was calculated.2)HepG2 cells were cultured in vitro.The cells were incubated with alkaloids(1.296、2.16、3.6、6、10)and non-alkaloids(0.25、0.5、1、2、4g·L-1)respectively.The cell activity was measured by MTT assay and IC50 was calculated.3)HepG2 cells were cultured in vitro,interfered by non-alkaloid(0.75、1、1.5、2 g·L-1)and alkaloid(1、2、6 g·L-1)sites.The cell membrane damage was measured by the instructions of LDH kit.The cell damage rate was calculated by measuring the absorbance of the sample.4)HepG2 cells were cultured in vitro and treated with non-alkaloid.The apoptosis was detected by flow cytometry at 12 h and 24 h respectively referring to the instructions of Annexin V-FITC/PI apoptosis detection kit.3.Toxicity study of RST and its extracts on mice by oral administration for 7 days1)Mice were given drugs repeatedly for 7 days.After administration,the mice were weighed.The blood samples were taken to measure the expression of UREAALT、AST、TB、CHE、CK in serum.The liver was removed and weighed,and the liver coefficient was calculated.2)Taking out the liver tissue and extracting the total RNAs from the tissue homogenate,determining the concentration,adjusting the concentration of RNA to the same level.And then RT-PCR were carried out to detect the expression of CYP1A2、CYP2E1 and CYP3A4 mRNA in mouse liver tissue.3)The protein was extracted from the tissue homogenate and the expression of CYP1A2、CYP2E1 and CYP3A4 protein was detected by Western Blot assay.Research result 1.Study on the antitoxic effect of mixed decoction1)Acute toxicity in mice.The LD50 of single decoction group was 19.623 g kg-1 and the mixed decoction group was 31.721 g kg-1(Calculation based on the amount of dose of RST).It is suggested that the compatibility has a attenuated effect.2)24 h after adminstration,LC50of embryonic zebrafish in the mixed decoction group of was significantly lower than that of single decoction group,and zebrafish embryos almost all died in the mixed decoction group.48 h after adminstration,there has shown the same result on the yolk sac stage zebrafish,.Results showed that the mixed decoction did not show the attenuation on the zebrafish model.3)The freeze-dried powder of single decoction,combined decoction and negative group all showed strong inhibitory effect on HepG2 cells at the concentration of 5 g·L-1 The inhibitory effects on HpeG2 cells of combined decoction and negative group were higher than those of single decoction.The results showed that the mixed decoction did not show the attenuation at the cellular level.However,when the concentration was 2.5-10 g·L-1,he inhibitory effect of combined decoction on HepG2 cells was higher than that of RST.However,when the concentration was 40 g·L-1,he inhibitory effect of RST on HepG2 cells was strongest.2.Study on the effects of different extraction parts of RSTThe alkaloids and non-alkaloids were separated,and non-alkaloids was identified as flavonoids and saponins by UPLC-Q-TOF-MS qualitative analysis.1 The non-alkaloid of the RST can delay the development of zebrafish.The LC50 of the non-alkaloid group was significantly lower than that of the alkaloid group(0.43 g·-1 vs 3.37 g·L-1）at 48h,and no death was found in the zebrafish embryos in the alkaloid group for 24 h.For zebrafish in yolk sac stage,the LC50 of non-alkaloid was lower than that of alkaloid,which was consistent with the toxic effect of zebrafish at embryonic stage.2)The IC50 of alkaloids and non-alkaloids to HepG2 cells were 9.044 and 0.983 g· L-1,rspectively.Which indicating that the toxicity of non-alkaloid was the strongest,and the toxicity of alkaloid sites was relatively weak.3)When the concentration of non-alkaloid was 2 g·L-1,the cell damage rate reached 67.31%,but the damage of alkaloid to HepG2 cell membrane was very weak at 6 g·L-1 concentration.The damage rate of alkaloid site to HepG2 cell membrane was lower than 10%.It is concluded that HepG2 cell is more sensitive to non-alkaloid.4)The total apoptosis rate of the blank group was 16.16±4.88%,and the total apoptosis rate of HepG2 cells treated with 1、2 g·L-1 of non-alkaloid for 24 h were 18.69±7.06%and 34.00±5.27%,respectively.The results showed that the non-alkaloid could induce the apoptosis of HepG2 cells in a dose-dependent manner,and when the drug concentration was 2 g-L-1,the total apoptotic rate was significantly different from that of the control group(P<0.05),and the apoptosis caused by non-alkaloid was mainly early apoptosis,which suggested that the drug might inhibit the cell vitality by inducing cell apoptosis.3.Toxicity study of RST and its extracts on mice by oral administration for 7 days1)On the 2ed、3rd、4th day of administration,the body weight of RST was significantly higher than that of the control group(P<0.05).Compared with the control group,the liver coefficient of the RST、alkaloid high-dose group and the non-alkaloid high-dose group was significantly different(P<0.05).2)ALT,AST,TB,UREA and CHE changed little in this study.The statistical results showed no significant difference.In the CK index test,the non alkaloid sites could significantly increase CK(P<0.05),and the rest of the groups could not significantly increase the expression of CK.3)After 7 days of continuous administration,compared with the control group,the mRNA of CYP450 in each group was increased in varying degrees,among which,the expression of CYP12 and CYP2E1 was significantly increased in the low and high dose groups of alkaloids(P<0.05).4)After 7 days of continuous administration,compared with the control group,the expression of CYP450 protein was increased to some extent in each group.The high dose of alkaloid could significantly increase the expression of CYP2E1(P<0.05),which showed the same trend as the corresponding expression of mRNA.It indicated that RST might cause hepatotoxicity by affecting the expression of CYP450 in liver tissue.ConclusionThe results of mouse acute-toxicity test showed that the compatibility of RST,RB and RC might reduce the toxicity.The results of cells’ toxicity suggested that the compatibility of RST,RB and RC might reduce the toxicity while the concentration was in a certain range.And alkaloids of RST might be the main toxic part.After administered continuously for 7 days,RST and its extracts can increased the liver index.The results of blood biochemistry test showed that high dose group of non-alkaloid could cause significant increase of CK.After administered continuously for 7 days,alkaloid of RST can cause mRNA(CYP1A2.CYP2E1 and CYP3A4)and protein expression increased in liver,suggesting alkaloid of RST might cause liver toxicity by affecting CYP450 enzymes.Non-alkaloid of RST might induce myocardial injury.In the acute toxicity test of zebrafish experiments,the mixed decoction showed more toxic.We speculate that the zebrafish toxicity experiment results were inconsistent and mouse toxicity test results may lie in:drug administration of zebrafish and clinical routine administration are in different ways,there may be different in metabolic mechanism.We focus on mouse toxicity test results,and we will continue to explore the toxicity mechanism of RST in the future research. |
PDF Full Text Request