| ObjectivePolygonum multiflorum is a traditional Chinese medicine tonic and belongs to the dry roots of Polygonum multiflorum Thunb.The taste is bitter,sweet and astringent,the nature is warmand attribute to liver and kidney.Its effects have replenishing blood,detoxification,cut off malaria,laxative.Modern pharmacological studies have shown its anti-aging,anti-inflammatory,liver protection and other effects.Traditionally,Heshouwu has never attracted attention because of its toxicity.However,in recent years,there have been more and more clinical case reports on the hepatotoxicity of Heshouwu.As a Chinese medicine known as “natural and safe”,its potential toxicity has also become domestic and international.At present,the chemical components and specific mechanism that cause liver toxicity of Polygonum multiflorum remain unclear.Based on this,this topic focuses on the active ingredients of Radix Polygoni multiflor that inducedpotential hepatotoxicity and possible mechanisms of its toxic effects.First,the normal rats were used to examine the chronic toxicity of 95% ethanol extract of Radix Polygoni multiflori,to confirm the toxic and toxic sites of Polygonum multiflorum.Then the chemical composition of Polygonum multiflorum was separated,and the toxicity of the isolated compound was screened by human normal liver cell line LO2 in vitro cell experiments.Through morphological observation,flow cytometry and Western blotting,the hepatotoxicity and apoptotic mechanisms of active ingredients derived from Polygonum multiflorum on rats and hepatocytes were studied.It revealed the biological mechanism of liver injury caused by Polygonum multiflorum,provided scientific data and reference for clinical rational use of Shouwu liver protection function,and provided clinicians with a new treatment idea and target.Methods 1.Study on the toxicity of chronic hepatic toxicity of 95% ethanol extract of Polygonum Multiflorum(1)The male rats with 150-180 g body weight were received vehicle control or Polygoni Multiflori Radix extracts(HSW-Ex)orally at 19.2(low dose),192(medium dose),or 1920 mg/kg/day(high dose),respectively,for 28 consecutive days.Signs of HSW-induced toxicity were monitored by clinical pathology and histopathology.(2)H&E were used for histological analysis.Incidence of hepatic lesions was determined by histologic analysis of the same portion of the liver from each animal.At the end of the 28 days,liver were removed,weighed,and fixed in 10% neutral buffered formalin,embedded in paraffin,sectioned at 6 ?m,and stained with H&E.(3)Analysis of serum liver function related indicators.At the end of the 28-day treatment,rat blood was collected and serum activities of ALT,AST,and ALP were quantified with a biochemical autoanalyser.(4)Measuring serum superoxide dismutase(SOD)activity.At the end of the 28 days treatment,liver were removed and stored at-80°C refrigerator.Then frozen liver tissues were quickly weighed prior to homogenization.The homogenates were then centrifuged and the supernatants were separated and enzyme activity was assessed.2.The efficacy of resveratrol and emodin derived from Polygonum multiflorum on the LO2 cells.(1)By the 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium bromide(MTT)assay,different concentrations(1.5625,3.125,6.25,12.5,25,50,100 and 200 ?g/mL)of resveratrol and emodin were used to evaluate the the efficacy of resveratrol and emodinon LO2 cells.(2)Flow cytometry(FCM)was used to investigate whether resveratrol and emodin resulted in apoptosis and the proportion of apoptotic cells.Q1,Q2,Q3,and Q4 denote necrotic,late apoptotic,viable(live),and early apoptotic regions.The sum of Q2 and Q4 regions represent cell apoptosis of LO2.(3)Ordinary light mirror;HE-Giemsa staining and Hoechst33258 by a phase contrast microscope and fluorescence microscopewere to illustrate cell morphological changes after the administration of resveratrol and emodin.And transmission electronic microscope(TEM)was used to observe the ultrastructural morphological changes in LO2 cells.(4)Western blot analysis was performed for resveratrol and emodin of apoptosis related proteins such as: Bax?Bcl-2?Caspase 3? Caspase 9 and TNF-?in LO2 cells.(5)Western blot analysis was performed for resveratrol and emodin of proteins such as: ERK,P-AKT,AKT and PI3 K in LO2 cells.(6)The data were analysed using SPSS Statistics version 16.0.Group means were compared by one-way analysis of variance(ANOVA)followed by Student-Newman-Keuls or Games-Howell Multiple Comparison Test.Differences with 95% confidence(p < 0.05)were considered significant.Results ?.Effect of active ingredients of Polygonum multiflorum on rat livers 1.HE results of rat livers.Compared to the vehicle control group,HSW-HD demonstrated severe hepatocellular degeneration along with necrotic areas while mild hepatocellular degeneration was observed with HSW-MD group.2.Effect of Polygonum multiflorum Thunb on serum ALT,AST and ALP in rats.Compared to the vehicle animals,serum ALT concentrations of the HSW-LD and HSW-MD group increased significantly(p<0.05)by 1.45 times,2.55 times and HSW-HD by 6.44 times(p<0.01);in contrast to ALT concentrations,serum AST increased significantly in the HSW-HD group by 2.41 times(p<0.01);Then serum ALP concentrations were significantly highly increased in the HSW-HD group(p<0.01)by 3.4 times.There were no significant differences in other groups,in comparison with the vehicle controls.3.Effect of Polygonum multiflorum Thunb on SOD in ratsThe animals treated with HSW-HD and HSW-MD showed a significant reduction by 2.56 times and 1.21 times(p<0.05),and HSW-LD decreased by 0.13 times in SOD activity but with no any significant difference when compared to the untreated vehicle control group.?.Effect of resveratrol and emodin,extracted from Polygonum multiflorum,on LO2 cells 1.The viability of LO2 was detected by MTT.The results showed that resveratrol and emodin improve LO2 proliferation at a low dose and showed a significant proliferation reduction a high dose,both in a dose-dependent manner.It suggests that the effect of resveratrol to LO2 cells depends on the dose and time.Resveratrol has an IC50 of 42.25-61.33 ?g/mL.2.Observation of the apoptotic cells in LO2 by flow cytometry(FCM).The sum of Q2+Q4 in the administration groups of resveratrol and emodin was significantly higher than that of the normal group.The Q2+Q4 proportion of apoptotic cells increased in a dose-dependent manner.3.Cell morphological changes of LO2 after the administration of resveratrol and emodin.(1)The result of ordinary light microscopy: compared with the control group,the cell morphology of resveratrol and emodin group were showing a long spindle shape,and the number of adherents was dose-dependently reduced.In the high concentration group,a large number of floating dead cells were seen,and the refractive index was poor.Giemsa staining showed that: compared with the control group,with the increase of emodin and resveratrol concentrations,the number of adherent cells was significantly reduced,the cell morphology was shrinking,and some of the nuclei were densely stained.(2)The results of fluorescent staining showed: dense nuclei and cytoplasm from resveratrol and emodin were densely stained,and massive blue fluorescence was observed;the higher the concentration,the smaller the number of cells.(3)After treatment with resveratrol of 100 ?g/mL for 24 h,TEM showed that nuclear chromatin condensation or cleavage,cell membrane blebbing and apoptotic bodies.Meanwhile,some autophagosomes,which had double layer membrane,were observed in the dose of 25-50 ?g/mL in LO2 cells.Altogether,these data demonstrated resveratrol induced LO2 cells apoptosis accompanied autophagy.The results of emodin 50 ?g/mL: there were some lysosomes in the cytoplasm and the formation of vacuoles.Integrated nuclear chromatin,mitochondrial bilayer membrane structures and autophagy-lysosome were observed.Cell necrosis also can be seen.The nuclear membrane and cytoplasm membrane showed broken and extinct,and there were lots of pieces nucleoplasm condensation and indistinct organelles.Group of Emodin 50 ?g/mL can be seen nothing structure change.4.Observation of apoptosis related proteinCaspase-3,Caspase-9,Bax,Bcl-2,and Bcl-2/Bax in LO2 cells after resveratrol and emodin treatment.(1)Western-bloting was used for dynamic observation of apoptosis related proteinCaspase-3 in LO2 cells.In normal group,Caspase-3<1,cell apoptosis is relatively little,While in model group treated with resveratrol of 50 ?g/mL,the content of Caspase-3 was increased(P<0.05).Therefore,occurrence of cell injury of LO2 may be related with excessive expression of Caspase-3.There was no significant difference between emodin groups and control group.(2)Western-bloting was used for dynamic observation of apoptosis related proteinsBax and Bcl-2 in LO2 cells.The results showed that,LO2 cells treated with resveratrol of 50 ?g/mL,Bax was continuously increased,and Bcl-2 was decreased,and Bax/Bcl-2 was also increased(P<0.05),compared with the control group.Abnormal level of Bcl-2/Bax was associated with the mechanism of cell damage caused by resveratrol.There was no significant difference between emodin groups and control group.(3)Western-bloting was used for dynamic observation of Caspase-9 in LO2 cells.Compared with the control group,there was no significant difference in both resveratrol and emodin groups.5.Observation of protein TNF-? in LO2 cells after resveratrol and emodin treatment.(1)Results showed: in LO2 cells treated with resveratrol of 50 and 100 ?g/mL,the content of TNF-? was obviouslyincreased(P<0.05),compared with control group.However,the content of TNF-? in LO2 cells treated by resveratrol of 25?g/mL,has no significance(NS),compared with other groups.The results illustrated cell injury induced by resveratrol 50 and 100 ?g/mL may be related to TNF-?.There was no significant difference between emodin groups and control group.6.Observation of proteincs ERK,P-AKT,AKTand PI3 K in LO2 cells after resveratrol and emodin treatment.(1)Compared with the control group,the expression of ERK in resveratrol 200?g/mL group was decreased(P<0.01).Compared with the control group,ERK expression was decreased in emodin 50,100 and 200 ?g/mL groups(P<0.05),and the difference was statistically significant.(2)Compared with the control group,the expression of P-AKT in resveratrol 50,100 and 200 ?g/mL groups was decreased(P<0.001).Compared with the control group,the expression of P-AKT in the emodin 50?g/mL group was increased(P<0.05);the expression of P-AKT in the emodin 200?g/mL group was decreased(P<0.05).(3)Compared with the control group,the expression of AKT in resveratrol 50,100 and 200 ?g/mL groups was decreased(P<0.05).The expression of AKT in emodin groups at 25,50,100 and 200 ?g/mL was decreased(P<0.05),and the difference was statistically significant.(4)Compared with the control group,the expression of PI3 K in resveratrol 50,100 and 200 ?g/mL groups was decreased(P<0.05).Compared with the control group,the expression of PI3 K in emodin 100 and 200 ?g/mL groups was decreased(P<0.05).Conclusion 1.The HSW-Ex may lead to liver damage,specifically at the high and medium dose(1920 mg/kg and 192 mg/kg,respectively),as indicated by the increased levels of ASP,ALT,and AST and decreased activity of SOD.2.(1)Resveratrol improved LO2 proliferation at1.5625-6.25 ?g/mL in a dose-dependent manner;cells treated with 12.5-100 ?g/mL showed a significant proliferation reduction dose-dependently,compared to control group.Meanwhile,the cells exhibited a smallest proliferation(36.09%)at the concentration 100?g/mL of resveratrol.The results showed that resveratrol had hepatotoxicity to LO2 cells.(2)The apotosis of LO2 cells may be related to the hepatotoxicity from resveratrol,accompanied with mitochondrial autophagy way.(3)Increased Caspase-3,Bax,Bax/Bcl-2,with decreased Bcl-2 in the LO2 cells,showed resveratrol induced damage to LO2 cells mainly by apoptotic pathways.The increase of TNF-? suggests that high concentrations of resveratrol have necrosis effect on LO2 cells.(4)Resveratrol reduced the expression of proteins that promote cell proliferation and inhibit apoptosis in LO2 cells,such as ERK,P-AKT,AKT,and PI3 K proteins,in a dose-dependent manner.It suggested that the mechanism of liver toxicity of resveratrol on LO2 cells might be related to the inhibition of the expression of ERK,P-AKT,AKT and PI3 K which inhibited apoptotic proteins.3.(1)Morphological and ultrastructural studies: emodin can cause apoptosis and necrosis in L-O2 cells,with mitochondrial autophagy,in a dose-dependent manner.(2)Emodin reduced the proliferation of LO2 cells and inhibited the expression of apoptotic proteins such as ERK,P-AKT,AKT and PI3 K in a dose-dependent manner.The mechanism of hepatotoxicity of emodin on LO2 cells may be related to the inhibition of the expression of ERK,P-AKT,AKT,and PI3 K that inhibit apoptosis. |
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