Effects Of Polygonum Multiflorum Thunb On The Uptake And Excretion Of Bilirubin And Hepatotoxicity Based On Cytokinesis

Objectives:To studied the effects of raw and processed Polygonum multiflorum Thunb and its main constituents on the hepatotoxicity,uptake and efflux of UCB by establishing the rat primary hepatocyte model.Estimated the cause and substance basis of Polygonum multiflorum Thunb,which is important for clinic to provided new theoretical basis and scientific support.Methods:1.The hepatotoxicity of the raw and processed Polygonum multiflorum Thunb and its main components in the hepatocytes.（1）Constructed the rat primary hepatocyte model,the primary hepatocyte morphology was observed under the microscope,and the hepatocyte yield and survival ratio were detected by trypan blue.（2）The RPME and PPME from 11 regions and 12 main constituents were administered,the toxicity determined by CCK-8 assay,estimating the region differences and constituents of Polygonum multiflorum Thunb hepatotoxicity.2.Effects of the raw and processed Polygonum multiflorum Thunb and its main constituents on the uptake of UCB in the hepatocytes（1）Constructed the detection methods of UCB in rat primary hepatocyte samples and sample processing methods.（2）Examined the effects of incubation time on the uptake of UCB in rat primary hepatocyte,to ascertain the optimal uptake time.（3）Investigated the effects of RPME and PPME from 11 regions and 12 constit-uents on the uptake of UCB in rat primary hepatocytes,estimating whether the jaundice related to Polygonum multiflorum Thunb and main constituents by influencing the uptake of UCB.Investigated the inhibiting type of Polygonum multiflorum Thunb and emodin,physcion gallic acid,catechin,epicatechin and rhein on the uptake of UCB in rat primary hepatocytes.3.Effects of raw and processed Polygonum multiflorum Thunb and its main constituents on the efflux of UCB in the hepatocytes.（1）Constructed the sandwich-cultured rat primary hepatocyte model.（2）Investigated the effects of RPME and PPME from 11 regions and 12 constit-uents on the efflux of UCB in rat primary hepatocytes,estimated whether the jaundice resulted from Polygonum multiflorum Thunb and main constituents to influence the efflux of UCB.Results:1.The hepatotoxicity of raw and processed Polygonum multiflorum Thunb and its main components in hepatocytes（1）With the increase of concentration,the hepatotoxicity of RPME and PPME from different regions became more and more powerful.At 12h,the minimum IC₅₀value of Shanxi was 1122±84.02μg/mL in RPME and Shandong was 4789±379.1μg/mL in PPME.At 24h,the minimum IC₅₀ value of Hubei was 1122±84.02μg/mL in RPME and Shanxi was 4789±379.1μg/mL in PPME.At 48h,the minimum IC₅₀ value of Henan was 1122±84.02μg/mL in RPME and Hubei was4789±379.1μg/mL in PPME.Compared with Guizhou,at 12h,there were respectively four producing areas with statistical differences in RPME and PPME;at 24h,there were four of RPME and one of PPME statistical difference;at 48 h,there was no statistical difference between all RPME and PPME producing areas.（2）With the increase of concentration,the hepatotoxicity of all constitu-ents became more and more powerful,the IC₅₀ values of Em,Em-G and Ep became smaller and smaller.At 12h,24h and 48h,IC₅₀ values of Em were:625±14.51,257.57±12.17,180.63±15.68μM;IC₅₀ values of Em-G were:785.4±91.09,657.77±7.51,462.3±43.3μM;IC₅₀ values of Ep were:422.07±26.1,380.33±30.42,341.3±15.68μM.Among them,the smallest IC₅₀ value of Ep was 422.07±26.1μM at 12h,and the IC₅₀ value of Ph was the lowest at 24h and 48h,which were 148.3±13.23μM and 176.93±6.56μM.Compared to Em,at 12h,24h,48h,there were respectively 6,7 and 9 constituents with statistical difference,2.Effects of raw and processed Polygonum multiflorum Thunb and its main constituents on the uptake of UCB 6in the hepatocytes（1）The detection method of UCB in primary hepatocytes was established with good specificity,the limit of quantification and detection were respectively 0.01μM and 0.005μM.UCB had a good linearity in the concentr-ation range of 0.01～2μM（R²=0.9998）.Intra-day precision and Inter-day precision RSD was<5.5%,the accuracy between 94.2%and 101.1%.It had good stability in repeated freezing and thawing and freezing process.The recovery rate of UCB in rat primary hepatocytes was>99.91%,with no matrix effect.（2）The uptake of UCB in rat primary hepatocytes is rapidly increased from10 to 40 min,then slowed down after 40 min and gradually reached a steady trend.（3）Gemfibrozilcould significantly inhibit the uptake of UCB,RPME from different areas had inhibitory effects on the uptake of UCB with the increase of concentration in rat primary hepatocytes.The IC₅₀ value of SX was the lowest,and the highest was Yunan,which were 52.54±3.33μg/mL and388.67±97.23μg/mL respectively.The IC₅₀ values of RPME in other localities had no significant difference with Guizhou.（4）PPME from different areas had inhibitory effects on the uptake of UCB with the increase of concentration in rat primary hepatocytes.The IC₅₀ value of Hubei was the lowest,and the highest is Shanxi.which were 204.2±13.72μg/mL and 440.67±39.64μg/mL respectively.There were statistically significant differences of Guangxi,Anhui,Sichuan,Yunan,Shanxi and Shanxi,which the IC₅₀ values of PPME in other localities were compared with GZ,and the inhibiting type was uncompetive inhibition.（5）All constituents had inhibitory effects on the uptake of UCB in ratprimary hepatocytes.The smallest IC₅₀ value was emodin,8.12±0.51μM,in addition to emodin,the IC₅₀ values of physcion,gallic acid,catechin,epicatechin and rhein are all about 20μM,which were 15.6±0.84μM,17.03±0.8μM,29.4±2.14μM,23.25±2.06μM and 23.97±2.02μM respectively.The results were statistically significant difference which compared to the IC₅₀value of emodin,the inhibiting type of all above was mixed-type inhibition.3.Effects of raw and processed Polygonum multiflorum Thunb and its main constituents on the efflux of UCB in the hepatocytes（1）The primary rat hepatocyte sandwich-cultured model was established well and fulfilled the requirement of UCB efflux experiment.（2）After incubation in standard buffer and Ca²⁺-free buffer,the efflux of UCB gradually reached a steady state at 10 min in rat primary hepatocytes.（3）In the presence of calcium,RPME of Henan and Guangxi（both 200μg/mL）,Hunan and Shanxi（both 16μg/mL）inhibited the accumulation of UCB,RPME of other producing areas or concentration had no significant or inductive effect on the UCB accumulation.Under the condition of calcium-free,RPME of He N and Guangxi（both 200μg/mL）,Henan（50μg/mL）,and Sichuan（16μg/mL）inhibited the accumulation of UCB,RPME of other producing areas or concentration had no significant or inductive effect on the UCB accumulation.In addition,except RPME from Hunan（200μg/mL）,Guizhou and Guangxi（both 50μg/mL）,and Henan（16μg/mL）inhibited the BEI of UCB,RPME of other producing areas or concentration had no significant or inductive effect on the BEI of UCB.For the CL of UCB,RPME from Hunan（16,200μg/mL）,Shanxi（16,50,200μg/mL）,and Guangxi（50,200μg/mL）were inhibition,RPME of other producing areas or concentration had no significant or inductive effect on the CL of UCB.（4）When calcium was present,PPME of Hubei（25,100,500μg/mL）and Shandong,Shanxi（both 500μg/mL）and Hunan（25,100μg/mL）had an inhibitory effect on the accumulation of UCB.PPME from other producing areas showed no significant or inductive effect on the accumulation of UCB.In the calcium-free state,PPME of all origins showed induction or no significant effect at the investigated concentrations.In addition,PPME from Hunan,He Nnan（both 25,100,500μg/mL）,Anhui and Shandong（both 25,100μg/mL）and Guangxi,Sichuan,Yunan（all 500μg/mL）inhibited the BEI of UCB,PPME of other localities or concentration showed induction or no significant effect.For the CL of UCB,PPME of Hunan and Henan（both 25,100,500μg/mL）,Anhui and Shandong（both 25,100μg/mL）,Shanxi and Yunan（both 500μg/mL）,Hubei（25μg/mL）showed inhibition,PPME from other localities or concentration had induction or no significant effect.（5）In the calcium-containing state,the five components of Em,A-Em,Ph,Ca,and Ep are at all investigated concentrations and Rh（20,100μM）,Ga（2.5,20μM）,and Ch,Ph-G（both 100μM）,Em-G（20μM）showed inhibitory effect on the accumulation of UCB,while other components and concentrations showed induction or no significant effect.In the calcium-free state,Ch and Ph are at all investigated concentrations and Em-G（20,100μM）and Ep,Ga,5-HMF（all20μM）,and Ph-G（2.5μM）showed inhibitory effect on the accumulation of UCB,other ingredients or concentrations showed induction or no significant effect.In addition,UCB BEI,Em and Ga（both 20,100μM）,Ca（2.5,100μM）,Epand Ph（both 100μM）were inhibitory effects,and other components or concentrations were inductive or no significant effect.For the CL of UCB,Ph,Ep,Ga（all 2.5,20,100μM）and A-Em（2.5,100μM）,Em and Ca（both2.5,100μM）,Em-G（2.5,20μM）,and Rh（100μM）and Ch（2.5μM）were all inhibitory effect,and other components or concentrations showed induction or no significant effect.conclusions:（1）The rat primary hepatocyte obtained by two-step collagenas eperfusion method has high vitality and enough to meet the requirements of experienments.（2）Contructed the adherent cultured and sandwich-cultured rat primary hepatocyte model active well,which can be better for hepatotoxicity,the uptake and efflux of UCB experiments.（3）The detection method of UCB in primary hepatocyte samples established in this experiment is of good specificity and high sensitivity,which can be used for the detection of UCB concentration in related samples.（4）RPME and PPME in all producing areas could inhibit the uptake of UCB and the type was uncompetitive inhibition.RPME and PPME in some areas inhibited the efflux of UCB.Therefore,the main reason for the jaundice related to inhibit the uptake of UCB resulted from Polygonum multiflorum Thunb in clinical,and the inhibitory effects were in different origins.In addition,there is indeed a attenuating effect in the processing,but at high doses,all areas of PPME still have hepatotoxicity.Therefore,using raw and processed Polygonum multiflorum Thunb which should be paid attention to its usage and differences in origin.The hepatotoxicity of Em,Ph,Ga,Ca,and Ep was obviously,and both UCB uptake and efflux were inhibited.Therefore,Em,Ph,Ga,Ca,and Ep may be the main components of Polygonum multiflorum Thunb in clinical practice.The specific mechanism needs further study.