Excretion Of Total Glucosides Of Paeony In Rats And Its Effect On Protein Expression Of CYP3A

Objective: Total glucosides of paeony (TGP) are the bioactive componentsextracted from white peony root, and the contents of paeoniflorin (Pae) andAlbiflorin (Alb) are the highest in TGP. Our group had compared thepharmacokinetic parameters of TGP in normal and CCl₄induced hepaticinjury rats, and found that the parameters were significantly different innormal and pathological state; meanwhile gastrointestinal absorption, plasmaprotein binding rate and tissue distribution characteristics of TGP had alsobeen studied. For further study of TGP pharmacokinetics, to clear theexcretion characteristics of TGP and study its effect on protein expression ofCYP3A, in our research we adopt HPLC to Simultaneous determination ofPae and Alb in rat urine, feces and bile as a means of solid-phase extractionmethod to clear the excretion pathway; in the meantime we adopt normal andCCl₄induced hepatic injury rats as experimental subjects, using Western Blotto observed its effect on protein expression of CYP3A, to explore the relatedmechanism of TGP hepatoprotective effect.Methods:1Study on the excretion of TGP in rats1.1Chromatographic conditionsThe HPLC assay was established by phenomenex C18column (4.6×250mm,5μm) with acetonitrile-0.1%acetic acid; the mobile phase proportion of theurine was16:84while the feces and bile were13:87; flow rate:1mL/min;detection wavelength:232nm; column temperature was25°C; injectionvolume:10μL.1.2The pretreatment of samples1.2.1The pretreatment of the urine0.4mL urine was precisely taken, then added distilled water0.2mL and20μL internal standard (IS) gentiopicrin solution, vortexed mixing, and extracted bythe activated solid phase extraction column, first with10%methanol2mLleaching of impurities, following the use of methanol1mL elution, the eluentvolume into1mL, centrifugation (12000r/min), the supernatant was gottenout and10μL was injected for the sample analysis.1.2.2The pretreatment of the fecesFecal samples were stored at-40℃for more than24h, freeze-dried,weighed and crushed. Then the fecal lyophilized powders were taken for0.01g, and added1mL distilled water, vortexed mixing5min, centrifuged (12000r/min) for10min, and the supernatant0.2mL was gotten out, added distilledwater0.2mL and20μL gentiopicrin solution, vortexed mixing, the rest dealwith the same pretreatment of the urine.1.2.3The pretreatment of the bile0.2mL bile was precisely taken, the remaining operating with "Thepretreatment of the urine".1.3Excretion1.3.1Excretion of TGP in rat urine and fecesWistar rats were5, set into the metabolic cage, given TGP aqueous solution(1.41g/kg) by gavage, and then collected the urine and feces, determined theconcentration of Pae and Alb in the samples by HPLC, according to theaccompanying standard curve to calculate the respective content.1.3.2Excretion of TGP in rat bileRats were given TGP aqueous solution (1.41g/kg) by gavage,25%urethaneby i.p, then collected bile by the separation of the common bile duct parallelintubation drainage of the bile duct, determined the concentration of Pae andAlb in the samples by HPLC, according to the accompanying standard curveto calculate the respective content.2Effect on protein expression of CYP3A by given TGP in normal rat25rats were randomly divided into five groups (normal control group, TGPlow, medium, high-dose group and positive control group). Normal controlgroup was treated with purified water, TGP low, medium, high-dose group rats were given TGP0.47,1.41,2.82g/kg, and14days in a row, meanwhilepositive control group was given dexamethasone75mg/kg for4days by i.p at11days. The rats were decapitated at15days. The livers were weighted tocalculate the liver coefficient. Liver microsomes were prepared by differentialcentrifugation and the protein expression of CYP3A was detected by WesternBlot.3Effect on protein expression of CYP3A by given TGP in CCl₄inducedhepatic injury rats30rats were randomly divided into six groups (normal control group, modelgroup, TGP low, medium, high-dose group and positive control group).Normal control group and model group were given purified water, TGP low,medium, high-dose groups were administered to give TGP0.47,1.41,2.82g/kg, positive control group rats were given DDB0.2g/kg, and14days in arow. All groups except normal control group were given50%CCl₄in corn oilto copy acute hepatic injury model by one-time subcutaneous injection, and allrats were decapitated after16h. Blood samples were collected for ALT andAST. The livers were weighted to calculate the liver coefficient. The lefthepatic lobe of the same parts were fixed in10%formaldehyde solution forroutine paraffin embedding, HE staining, and then observed the tissue injuryby light microscopy. Parts of the liver tissue were prepared for livermicrosomes by differential centrifugation and the protein expression ofCYP3A was detected by Western Blot.Results:1Study on the excretion of TGP in rats1.1Methodology surveyPae, Alb and IS peaks had good specificity and negative interference in therat urine, feces and bile conditions. The intra-day and inter-day RSD were lessthan10%, accuracy90%～110%. The absolute recovery of Pae, Alb and ISwere all more than90%. The samples after pretreatment could keep steadyafter room temperature and freeze-thaw cycle. 1.2Excretion of TGP in rat urine, feces and bileRats were given1.41g/kg TGP, and the cumulative excretions of Pae inurine and feces in48h and bile in36h were (610.0±53.9),(60630.4±4546.3),(5640.3±504.3) μg, the cumulative excretion percentages were (0.583±0.042)%,(59.11±6.87)%,(4.88±0.43)%. The cumulative excretions of Alb were(158.0±21.1),(27315.1±2383.4),(2462.1±211.2) μg, the cumulative excretionpercentages were (0.424±0.056)%,(74.70±9.17)%and (5.98±0.48)%.2Effect on protein expression of CYP3A by given TGP in normal ratCompared with normal control group, positive control group givendexamethasone showed a significant induction of CYP3A protein expression(P<0.01) increased by140%than the normal control group; TGP low andmiddle dose groups tended to increase compared with control group,respectively, increased by9%,19%, but no significant difference (P>0.05);TGP high dose group was significantly induced CYP3A protein expressionincreased by44%than the normal control group (P<0.01).3Effect on protein expression of CYP3A by given TGP in CCl₄inducedhepatic injury rats3.1Effect on liver coefficients of CCl₄induced hepatic injury rats by givenTGPCompared with the normal control group, the liver coefficients of the modelgroup ware significantly increased, significantly higher than normal controlrats (P<0.01). The pretreatment by TGP and DDB can significantly reduce theliver coefficients increased caused by CCl₄induced hepatic injury (P<0.05orP<0.01).3.2Effect on liver function of CCl₄induced hepatic injury rats by given TGPCompared with normal control group, ALT and AST of model group weresignificantly increased (P<0.01). Compared with model group, pretreated withTGP or DDB, can significantly reduce the levels of ALT and AST (P<0.05orP<0.01), and the TGP high-dose group and the DDB group efficacy wassignificantly better than TGP low and medium-dose group.3.3The pathological observation of liverThe observation of rats in normal control group by the naked eye: normal liver morphology, color red, neat edge, a soft texture; microscope showednormal lobular architecture, liver cells arranged in neat rows and the nuclei ofmorphologically normal, liver cell cord legible, no swelling, degeneration,necrosis, inflammatory cell infiltration and so on. Model group by the nakedeye: the enlargement of the liver volume, khaki gray, the edge of a dull, brittle;Microscopically, the hepatic lobule of normal structure was destroyed, thestructural disorder of the liver tissue, the cell state was unclear and bubble-likechange, cell cord dissociation, liver cells swelling, and diffuse inflammatorycell infiltration; each TGP group and DDB group could alleviate theabove-mentioned pathological changes.3.4Effect on protein expression of CYP3A by given TGP in CCl₄inducedhepatic injury ratsCompared with normal control group, model group showed a significantinduction of CYP3A protein expression (P<0.01) decreased by31%than thenormal control group. Positive control group was given DDB significantlyinduced protein expression of CYP3A, than hepatic injury group to improve59%(P<0.01); There was a marked induction of CYP3A protein expression inTGP low, medium, high-dose group, respectively as compared to the CCl₄hepatic injury group increased by16%(P<0.01),30%(P<0.01),52%(P<0.01).Conclusion:1Rats were given1.41g/kg TGP, the cumulative excretions of Pae in theadministration of the amount in urine, feces and bile were (0.583±0.042)%,(59.11±6.87)%and (4.88±0.43)%, respectively; and Alb were (0.424±0.056)%,(74.70±9.17)%and (5.98±0.48)%. It showed that TGP mainly via bileexcretion in vivo.2Normal rats were treated with TGP of low, medium, high dosages (0.47,1.41,2.82g/kg) for14days, then liver microsomes were prepared after24h,and the protein expression of CYP3A was detected by Western Blot. TGP lowand medium dose groups tended to increase, but no statistical significancecompared with control group. High-dose of TGP group was significantly induced CYP3A protein expression increased by44%compared with normalcontrol group. It suggested that this might be involved in hepatoprotectivemechanisms of TGP.3It was found that CCl₄can significantly reduce the level of rat CYP3Aprotein expression, decreased by31%compared with normal control group,suggesting that CYP3A protein expression levels may be closely related toliver injury. Rats were given low, medium and high dose TGP for14days,which can significantly improve the CCl₄-induced acute liver injury, asignificant increase CYP3A protein levels, suggesting that this might be oneof the mechanisms of TGP treatment liver injury.