Study On The Active Constituents,anti-cholestasis Effect And Mechanism Of Rhodiola Crenulata

Farnesoid X Receptor（FXR）is one of the most important nuclear receptors to maintain bile acid enterohepatic circulation and homeostasis.When the synthesis or secretion of bile acids is interfered,toxic bile acids remain in the liver and then cause liver damages.Rhodiola crenulata（HK.F.Et Thoms.）H.Ohba is a genuine source plant of Rhodiola medicinal materials included in the Chinese Pharmacopoeia with the.effectiveness of invigorating qi,promoting blood circulation and relieving pulse and asthma.Pharmacological studies have found that it plays an important role in liver protection,neuroprotection and anti-tumor effects.Previoulsy study indicated that the compound from Rhodiola crenulata exhibieted hepatoprotective effects on obstructive jaundice caused by obstruction of bile acid excretion,but whether it can activate FXR to regulate bile acids to exert an anti-cholestasis effect is not clear.Therefore,in order to determine whether the active ingredients of Rhodiola crenulata have an anti-intrahepatic cholestasis and its mechanism,this paper carried out the following three aspects of work:1.Study on the active ingredients of Rhodiola crenulata;2.Study of the active ingredient on the pharmacodynamics and mechanism of anti-intrahepatic cholestasis in vivo;3.Study of the active ingredient on the mechanism of FXR activated in vitro.1.Study on the active ingredients of Rhodiola crenulata with FXR agonistic effects.（1）Silica column,Sephedex LH-20,semi-preparative HPLC and preparative TLC were used to separate chemical components,and 12 compounds were identified by ¹H-NMR and ¹³C-NMR techniques.These compounds were crenulatin（QSY-1）,tyrosol（QSY-2）,p-cresol（QSY-3）,uridine（QSY-4）,salidroside（QSY-5）,kaempferol（QSY-6）,prinsepiol（QSY-7）,caffeic acid（QSY-8）,2,6-dimethoxyacetophenone-4-O-β-D-glucose（QSY-9）,p-hydroxybenzoic acid（QSY-10）,kenposide A（QSY-11）,4,5-dicaffeoylquinate（QSY-12）.Among them,QSY-3,QSY-7 and QSY-12 were isolated from genus Rhodiola L.for the first time,and QSY-9 was isolated from this plant for the first time.（2）The FXR was inhibited by guggulsterone in L-02 cells.The immunofluorescence technique was used to screen the activities of all isolated compounds.The results showed that tyrosol,salidroside,kaempferol,prinsepiol and 2,6-dimethoxyacetophenone-4-O-β-D-glucose could up-regulate the expression of FXR in guggulsterone-induced cells,indicating their potential anti-cholestasis effect by activating FXR.Since salidroside is the main active component of Rhodiola crenulata,this compound was selected for the follow-up research.2.Study on the protective effect and mechanism of salidroside against alpha-naphthylisothi（ANIT）-induced intrahepatic cholestasis in rats.The SD rats were divided into six groups including normal group,model group（60 mg/kg ANIT）,positive control group（100 mg/kg UDCA）,salidroside low-dose group（10 mg/kg,SAL-L）,medium-dose group（30 mg/kg,SAL-M）and high-dose group（60 mg/kg,SAL-H）.Rats in the positive control and salidroside groups were administered by intragastric administration for 7 days respectively.On the 5th day,apart from the normal group,the rats in the other groups were administered with ANIT olive oil solution（60mg/kg）.（1）The effect of salidroside on the treatment of intrahepatic cholestasis was evaluated of the bile flow rate,serum biochemical indicators,liver tissue pathological sections and inflammatory factors.The results showed that salidroside improved intrahepatic cholestasis in rats by ways of increasing the bile flow,decreasing serum biochemical indicators as ALT,ALP,AST,GGT,TBIL,DBIL and TBA,attenuating liver histopathological injuries and regulating inflammatory factors as TNF-α,IL-1β,IL-6 and IL-10.（2）FXR and its downstream genes were detected by western blotting to study the mechanism of salidroside against intrahepatic cholestasis in rats.Salidroside could up-regulate the protein expression of FXR,SHP1,SHP2,MRP2,BSEP and NTCP,while down-regulate the expressions of CYP7A1 and CYP27A1.The above results indicated that salidroside might protect against intrahepatic cholestasis through the activation of FXR to regulate bile acid synthesis and transport.3.Study on the mechanisms of salidroside via FXR in vitro.L-02 cells with different treatments were given salidroside low,medium and high doses（20,40 and 80μmol/L）and positive drug GW4064（6μmol/L）,respectively.The expression of FXR and its downstream genes were detected using immunofluorescence technology,reverse transcription-polymerase chain reaction（RT-PCR）and western blot methods.（1）Effects of salidroside on FXR and downstream genes after guggulsterone inhibited FXR expression.Salidroside could reverse the inhibitory effect on FXR,and up-regulate the expression of SHP1,SHP2,MRP2,BSEP and NTCP while down-regulate the expression of CYP7A1 and CYP27A1.This result was consistent with the effect of salidroside on anti-cholestasis in rats.（2）Effects of salidroside on FXR and downstream genes after knockdown of FXR expression was interfered by si RNA.When FXR expressopion was silenced,the expressions of downstream genes were also affected.Salidroside could activate FXR,and then up-regulate the expression of SHP1,SHP2,MRP2,BSEP and NTCP while down-regulate the expression of CYP7A1 and CYP27A1 at both m RNA and protein levels.（3）Effects of salidroside on FXR and downstream genes after overexpression plasmid highly upregulated FXR.expression Salidroside could further promote the activation effect of plasmid on FXR,and regulate the downstream genes related to bile acid synthesis and transport which were based on FXR.In conlusion,12 compounds were isolated and identified from Rhodiola crenulata,and 5 of them showed FXR activating effects including salidroside.Salidroside exhibited significant anti-intrahepatic cholestasis activity in vivo and in vitro by activating FXR and then regulation of its downstream genes as SHP1,SHP2,CYP7A1,CYP27A1,MRP2,BSEP and NTCP.This study laid the foundation for the development of FXR target-based drugs and their lead compounds,and for the further utilization of this medicinal materials.