This study was conducted to investigate the role of silibinin in the change of biochemical indices of rabbits poisoned with of amanita virosa to provide the experimental evidence on the ability of hepatocellular protection of silibinin against toxic effect of amatoxin.
Journal of military pharmaco-medicine n06-2018 THE EFFECTIVENESS OF SILIBININ ON THE CHANGE IN SOME BLOOD BIOCHEMICAL INDICES AND LIVER HISTOPATHOLOGY OF RABBIT EXPERIMENTALLY POISONED WITH AMANITA VIROSA MUSHROOM Ngo Thi Thanh Hai*; Nguyen Thanh Binh*; Tran Van Tung*; Be Hong Thu** SUMMARY Objectives: To evaluate the effectiveness of silibinin on the change in some blood biochemical indices and liver histopathology of rabbit poisoned with Amanita virosa mushroom in order to get evidence of protective effect of the drug against the toxicity of amatoxin Subjects and methods: Rabbits were randomly grouped then poisoned with Amanita virosa mushroom 12 hour after the intoxication, the rabbits were given silibinin then blood samples were collected for the testing of GOT, GPT, GGT, urea, glucose, total bilirubin and direct bilirubin on a automated system Liver histology was processed and examined followed standard HE staining procedure Results: GOT, GPT, GGT concentrations in blood of rabbits poisoned with Amanita rd th virosa in the group with silibinin treatment decreased on the and days after poisoning compared to the group without treatment Silibinin treatment also decreased hepatocellular damages caused by Amanita virosa mushroom poisoning Conclusion: Silibinin has protective effect against the toxicity of Amanita virosa mushroom * Keywords: Silibinin; Amatoxin; Amanita virosa; Blood biochemical indices; Liver histopathology; Rabbit INTRODUCTION According to statistics from the Poison Control Center of Vietnam Military Medical University from 2004 to 2011, in Backan province there were 28 incidences of mushroom poisoning with the total number of 94 infected people and 14 deaths The results of investigation showed that the deaths from poisoning of poisonous mushrooms in Backan province were caused by Amanita virosa [1] Amanita virosa is pure white, fleshy, very beautiful and attractive Amanita virosa contains amanitines that cause slow and sustainable poisoning to heat and has a very high toxicity Characteristics of these toxins are to cause hepatocellular necrosis leading to liver failure and death [3, 4, 5] The principle of treatment Amanita virosa poisoning includes the limited absorption and increased excretion of amatoxin, and use liver protection drugs as soon as possible accompanied by maintaining vital functions and symptomatic treatment [5, 6] * Vietnam Military Medical University ** Bachmai Hospital Corresponding author: Ngo Thi Thanh Hai (ngohaixpnoi@gmail.com) Date received: 18/05/2018 Date accepted: 20/06/2018 146 Journal of military pharmaco-medicine n06-2018 Silibinin dihemisusccinate prevents amatoxin from entering hepatocytes, reducing amatoxin in the intestinal and hepatic circulation, thereby increasing amatoxin excretion in bile and increasing the synthesis of ARN polymerase to reduce hepatocellular injury [4 ,6, 7] It is recommended that this drug should be used as an antidote to amantoxin This study was conducted to: Investigate the role of silibinin in the change of biochemical indices of rabbits poisoned with of amanita virosa to provide the experimental evidence on the ability of hepatocellular protection of silibinin against toxic effect of amatoxin SUBJECTS AND METHODS Subjects - Amanita virosa mushroom samples were collected in Backan province After collection, the mushroom samples were weighed, then preserved in ethanol till next experiment - Rabbit: 30 rabbits, regardless of male or female, healthy, weight: 2.0 ± 0.2 kg The rabbits were raised in the same mode during the experimental period ® - Legalon SIL (silibinin dihemisusccinate) 528.5 mg Research methods * Method of poisoning on rabbits: Amanita virosa preserved samples were processed to evaporate ethanol The ethanol free samples were then homogenized with a tissue homogenizer Water was then added to dilute the sample then filtered with a filter paper The homogenizing and filtering procedure were repeated three times to remove fiber and particulate material from the mushroom The filtered solution was then administered orally into rabbit stomach by specialized equipment with a dosage equal to 2/3 of the minimum lethal dose (LDmin) which was pre-determined * Method of conducting the indicators: - 30 rabbits were divided into groups, each group had 10 rabbits marked separately - Collect blood from vein in rabbit ears before and after poisoning, on the first, third and fifth days to test biochemical and hematological indicators - Group is not treated by drug after poisoning - Group 3: After poisoning day, inject silibinin into rabbit vein with a maintenance dose of 60 mg/kg/day for days - Biochemical and hematological indicators include: GOT, GPT, GGT, and urea concentrations; creatinine, electrolytes, blood sugar, total and bilirubin, etc are directly performed on automatic biochemical analyzer - Liver histology was processed and examined followed standard HE staining procedure at Department of Pathology, 103 Military Hospital - Evaluation of clinical criteria: Skip meals, diarrhea, mischievous level, etc * Statistical processing method: The data is averaged (X), standard deviation (SD) and compare average values Calculate p before and after poisoning, compare between the experimental group and control group by t-test 147 Journal of military pharmaco-medicine n06-2018 RESULTS Changes in GOT, GPT and GGT activities in rabbits poisoned with Aminata virosa treated or untreated with silibinin Group UI/L Group 800 Group 748.51 700 600 500 351.27 400 300 200 100 44.29 51.48 49.93 42.9 44.02 153.71 47.72 182.13 46.79 Time 43.21 Before poisoning After 12h 3rd days after 5th days after Figure 1: Changes in GOT activity GOT activity in serum of rabbits poisoned with Aminata virosa increased highly on the 3rd and 5th days after poisoning However, GOT activity in serum of poisoned rabbits untreated with sibilinin was higher than that in poisoned rabbits treated with sibilinin (p < 0.05) Group UI/L Group Group 700 598.33 600 500 333.75 400 300 200 100 54.56 60.32 62.94 59.19 63.1 70.39 101.64 66.3 54.14 Before poisoning 181.42 After 12h 3rd days after 5th days after Time Figure 2: Changes in GPT activity GPT activity in serum of rabbits poisoned with Aminata virosa increased highly on the 3rd and 5th days after poisoning However, GPT activity in serum of rabbits untreated with sibilinin was higher than that of rabbits treated with sibilinin (p < 0.05) 148 Journal of military pharmaco-medicine n06-2018 Before poisoning UI/L After 12h 3rd days after 192.5 5th days after 200 180 167.14 160 138.83 140 126.05 120 100 80 44.05 60 40 24.29 27.86 30.29 11.46 11.49 20 12.31 11.68 Group Group Group Group Figure 3: Changes in GGT activity in groups GGT activity in non-poisoned groups did not differ between time points In the untreated group (group 2), GGT activity increased significantly from day In the treated group (group 3), GGT activity also increased from day after poisoning However, GGT activity in group was significantly lower than group Changes on CPK, glucose and urea levels in serum of rabbits poisoned with Aminata virosa treated and untreated with silibinin UI/L Group Group Group 1400 1167 1200 1000 716.8 800 600 266 400 200 635.5 287.8 284.8 271.4 491.7 Before poisoning After 12h 3rd days after 5th days after Figure 4: Changes in CPK level in groups Before poisoning and after poisoning day, CPK level did not differ between groups with p > 0.05 CPK level increased at days after poisoning and CPK in group increased significantly higher than group with p < 0.05 149 Journal of military pharmaco-medicine n06-2018 mmol/L Before poisoning After 12h 7.6 3rd days after 7.2 7.1 6.5 6.3 6.1 5.7 6.2 5th days after 6.1 6.4 Group Group Group Group Figure 5: Changes in glucose level Average blood glucose level in groups and increased slightly at 12 hours after poisoning compared with before poisoning Plasma glucose concentrations on days and after poisoning did not differ between three groups mmol/L Group Group Group Before poisoning After 12h 3rd days after 5th days after Time Figure 6: Changes in urea concentration The plasma concentrations of uremia in rabbit blood at the time before and after poisoning were not statistically significant 150 Journal of military pharmaco-medicine n06-2018 Changes in liver histopathology of rabbits poisoned with Aminata virosa treated and untreated with silibinin A B C Figure 7: Liver histopathology of rabbit normal (A, H.Ex.400), rabbit poisoned with Amanita virosa untreated with silibinin (B, H.Ex.400), rabbit poisoned with Amanita virosa treated with silibinin (C, H.Ex400) (The liver of rabbit poisoned with Aminata virosa untreated with silibilin (B) showed acute lesions: The central vein and vasculature in the liver were swollen, enlarged, hepatocellular degeneration and necrosis The infection by polymorphonuclear leukocytes and lymphocytes into the portal and hepatic lobules The liver of rabbit poisoned with Aminata virosa used intravenous silibinin (C) showed a significant reduction in edema and necrosis and moderate hepatocellular degeneration) DISCUSSION The effect of Amanita virosa on some indicators of liver function AST and ALT are the most abundant enzymes in the hepatocytes and play the role in transporting amines in cell metabolism AST and ALT activities are usually relatively stable in cytoplasm of hepatocytes In hepatocytes, AST is mostly in cytoplasm and about 35 - 40% in plastid, while ALT is only in the cytoplasm When the hepatocytes are injured, the permeability of the cell membrane changes, causing AST and ALT escape from cytoplasm into blood, resulting AST and ALT of serum increase The more severe the hepatocytes are injured, the more the AST, ALT activities in serum increase, especially in cases of hepatocellular necrosis γ-GT is an enzyme of hepatocytes The activity of this enzyme increases along with hepatocellular injury, especially the injury caused by poisoning The research results showed that: AST, ALT, γ-GT concentrations in serum of rabbits poisoned with Amanita virosa increased significantly on the 3rd and 5th days compared to those before poisoning, and AST activity increased higher than ALT activity This proved that the hepatocytes were severely injured with hepatocellular necrosis In the event of hepatocellular necrosis, the plastid is destroyed causing 151 Journal of military pharmaco-medicine n06-2018 AST from plastid escape into blood, plus AST contained in cytoplasm leading AST in serum increase higher than ALT Our research results were also consistent with foreign authors’, that the liver of patients poisoned with mushrooms containing amatoxin such as Amanita virosa, Amanita verna, Amanita phaloides, etc is usually injured severely Patients with hepatic failure due to poisonous mushrooms are mostly fatal and these patients can only be rescued thank to liver transplantation Hoang Cong Minh’s research (2009) on rabbits poisoned with Amanita verna containing amatoxin showed that AST activity on the 5th day increased 25 times, ALT activity increased 12 times higher than before poisoning [2] According to Tamas R Peredy (2015), silibinin that is given intravenously was effective in preventing liver injury and should be used as soon as possible When silibinin is not available for intravenous delivery, it should drain gallbladder through the skin The results in the group treated by intravenous silibinin on the 3rd and 5th days after poisoning, GOT, GPT and GGT concentrations decreased markedly with p < 0.005 The effect of Amanita virosa on urea concentration in rabbit blood The research results showed that the urea concentration in rabbits poisoned with Amanita virosa changed insignificantly compared to before poisoning According to Tamas (2015) and Timothy (2015), the toxins of Amanita virosa in later stage will cause renal cell injury Urea is the major degredation product of protein and synthesized in the liver Creatinine is a 152 decomposition product of phosphocreatinerich organisms, especially in muscle fibers Urea and creatinine are excreted through the kidney and by monitoring the concentration of these two substances in blood, we can assess the functions and level of kidney injury A number of foreign authors studying the effects of amatoxin on animals also found that amatoxin causes damage to the renal tubules In this research, urea concentration did not change much at the research times, probably because the rabbits were only poisoned with a dosage equal to 2/3 of the minimum and determined lethal dose The effect of Amanita virosa on glucose metabolism function Glucose is the primary energy source of organs, especially brain and muscles Glucose in blood is one of the indicators to assess the glucose metabolism in the body The research results showed that glucose concentration in blood increased slightly on the first day and was changed unclearly on the days after poisoning and there was no difference in groups with and without treatment Research results of Floersheim G.L (1987); Chang A.K (2007); Bivins et al (1985) showed that glucose concentration in blood decreased in cases of poisoning with species of mushroom containing amatoxin, and in some cases, blood glucose dropped to very low The effect of Amanita virosa on CPK concentration in blood of poisoned rabbit CPK usually increases in cases of severe poisoning and acute destruction of striated muscle cells In this research, CPK concentration increased significantly Journal of military pharmaco-medicine n06-2018 on the 3rd and 5th days after poisoning with p < 0.05 After treatment with silibinin, CPK concentration decreased significantly in group being treated with silibinin compared to the group without treatment Changes on liver histopathology of rabbits poisoned with Aminata virosa treated and untreated with silibinin Liver of rabbit poisoned with Aminata virosa untreated (figure 7A) showed acute lesions: The central vein and vasculature in the liver were swollen, enlarged, hepatocellular degeneration and necrosis In contrast, after treatment by intravenous injection of silibinin, we found the significant reduction in degree of edema, necrosis and moderate hepatocellular degeneration in the liver of rabbit poisoned with Aminata virosa (figure 7B) CONCLUSION The blood biochemical indices and liver histopathology suggest that silibinin has protective effect against the toxicity of Amanita virosa mushroom REFERENCES Ngo Thi Thanh Hai, Hoang Cong Minh, Be Hong Thu Situation of poisoning of poisonous mushrooms in Backan province for years (2004 - 2011) Journal of Military Pharmaco-Medicine 2012, 37 (7), pp.89-93 Hoang Cong Minh Study the effects of Amanita verna extract on some biochemical indicators in rabbits Practical Medicine Magazine 2009, (656), pp.14-16 Trinh Tam Kiet List of large mushrooms in Vietnam Hanoi Agriculture Publishing House 1996, pp.62-80 Tamas R Peredy Amatoxin-containing mushroom poisoning including ingestion of Amanita phalloides Uptodate 2015 Timothy J Wiegand Clinical manifestations and evaluation of mushroom poisoning Uptodate 2015 New Zealand National Poisons Centre Amatoxin 2015 Linsay Murray Approaches to mushrooms poisoning Toxicology Handbook Second edition, Churchill Livingstone 2011, pp.44-49 153 Journal of military pharmaco-medicine n06-2018 CURRENT SITUATION OF HEALTHCARE RESOURCES AT COMMUNE LEVEL IN BORDER AREAS OF TAY NGUYEN FROM THE YEAR 2014 - 2016 Nguyen Minh Hung*; Trinh Thanh Hung*; Nguyen Van Chuyen** Nguyen Van Ba***; Nguyen Dinh Thanh***; Le Bach Quang** SUMMARY Objectives: To study the status of healthcare resources at commune level in border areas of Tay Nguyen (2014 - 2016) Subject and methods: A cross-sectional study, along with data retrospective method to describe healthcare situation in 28 commune-level health stations border areas of Tay Nguyen Results: Commune health system have enough health staff; however, they lack a structure of professional qualifications 92.86% of commune health system have doctors, of which 88.46% were general practitioners, only 3.85% were traditional medicine doctors and 7.69% were pediatricians The percentage of unskilled doctors remained high, accounting for 57.69% All commune health system had their own facilities; most of them did not have enough functional departments as prescribed Their equipment was still limited compared to regualtions, especially basic equipment for medical examination and special treatment Conclusion: There was a lack of human resources and facilities in healthcare systems at commune level in border areas of Tay Nguyen * Keywords: Healthcare resources; Commune health system; Border areas; Tay Nguyen INTRODUCTION There are 28 communes and 12 districts of Kontum, Gialai, Daklak, Daknong sharing a border with Laos, Cambodia, including 530 km of border line, of which Laos has 142 km, Cambodia has 388 km This area has the lowest socioeconomic status and the poorest transportation system in Tay Nguyen Health care for people in the border area of Tay Nguyen is still heavily dependent on grassroots health care, especially at commune and village levels However, this area encounters many difficulties in human resources, medical infrastructure and equipment The medical management in some aspects is limited; the quality of health care is still inadequate Therefore, many health indicators such as health care services and others of Tay Nguyen are slowly improved compared with other regions and with general level of the country Hence, a research on the status of health resources at the commune level in Tay Nguyen is very necessary This is a scientific basis to develop solutions to improve the medical examination and treatment capacity of communes Objectives: Research on the status of healthcare resources at commune level in border areas of Tay Nguyen (2014 - 2016) * Ministry of Science and Technology ** Vietnam Military Medical University *** 103 Military Hospital Corresponding author: Nguyen Minh Hung (hungnm@most.gov.vn) Date received: 10/04/2018 Date accepted: 20/06/2018 154 ... study was conducted to: Investigate the role of silibinin in the change of biochemical indices of rabbits poisoned with of amanita virosa to provide the experimental evidence on the ability of hepatocellular... poisoning with species of mushroom containing amatoxin, and in some cases, blood glucose dropped to very low The effect of Amanita virosa on CPK concentration in blood of poisoned rabbit CPK usually increases... silibinin (B, H.Ex.400), rabbit poisoned with Amanita virosa treated with silibinin (C, H.Ex400) (The liver of rabbit poisoned with Aminata virosa untreated with silibilin (B) showed acute lesions: