Comparative pharmacodynamic study of aqueous extract of Rehmanniae Radix Praeparata and drying Rehmannia root for amelioration of hepatic ischemia-reperfusion injury by inhibiting ferroptosis process
Objective Based on comparing the efficacy and mechanism of aqueous extracts of Rehmanniae Radix Praeparata(RRPAE)and drying Rehmannia root(DRR)for the treatment of hepatic ischemia-reperfusion injury(HIRI),to provide a better therapeutic option for clinical patients.Method Totally 48 mice were randomly divided into eight groups:the sham group,the model group,the low,medium,and high dose groups of RRPAE(2.5,5.0 and 10.0 g·kg-1),and the low,medium,and high dose groups of DRRAE(2.5,5.0 and 10.0 g·kg-1),with six mice in each group.After one week of pre-administration of different doses of RRPAE or DRRAE by ig,the mouse HIRI model was established,and the HE staining was used to observe the histopathological changes in the livers of the mice in each group.The kits were used to detect the levels of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),lactate dehydrogenase(LDH),nitric oxide(NO)levels.The kits were used to detect mouse liver superoxide dismutase(SOD),nitric oxide(NO),and malondialdehyde(MDA)levels.Western blotting was used to detect ferroptosis-associated proteins in mouse liver tissues of each group:Transferrin,solute carrier family 7 member 11(SLC7A11),solute carrier family 39 member 14(SLC39A14),and poly rC binding protein 2(PCBP2).After culturing and treating BRL hepatocytes with monocrotaline(MCT,500 μmol·L-1)or ferroptosis agonist erastin(10 μmol·L-1),different doses of RRPAE(50,100,and 150 μgmL-1)were administered for 24 h,and then the cellular ferrous iron/total iron ions and glutathione content were detected.Results Compared with the model group,after pre-administration of RRPAE or DRRAE,the liver and spleen coefficients of mice in the treatment group showed a downward trend,with significant differences in liver coefficients in the low and high dose RRPAE groups(P＜0.05,0.001).HE staining results showed significant ischemia-reperfusion region and hepatocellular necrosis in the HIRI group,and the hepatocellular status was recovered after both RRPAE and DRRAE administration,and the former was significantly better than the latter.The biochemical results showed that compared with the model group,the serum levels of AST,ALT and LDH,which were indicators of liver injury,decreased significantly,while the level of NO increased significantly;the level of hepatic MDA decreased significantly,while the levels of SOD and NO increased significantly(P＜0.05,0.01),and the modulation effect of DRR on the above indicators of liver injury and oxidative stress was not significant.The results of Western blotting experiments showed that the RRPAE was able to significantly inhibit the expression of Transferrin and SLC39A14-related ferroptosis-promoting proteins,and down-regulate the expression of SLC7A11 and PCBP2-related ferroptosis-suppressing proteins(P＜0.05,0.001),whereas the DRRAE did not significantly regulate the above iron-death-related proteins.At the same time,RRPAE directly inhibited the ferroptosis process of hepatocytes induced by monocrotaline or ferroptosis agonist erastin,which was accompanied by an increase in ferrous iron/total iron ions and glutathione content in the cell culture medium(P＜0.05,0.01,and 0.001).Conclusion The medicinal type of DRR with better effect in alleviating hepatic ischemia-reperfusion injury is RRPAE,and its mechanism of action may be related to the regulation of Transferrin,SLC7A11,SLC39A14,and PCBP2 protein expression and inhibition of ferroptosis process in mice.
aqueous extract of Rehmanniae Radix Praeparataaqueous extract of drying Rehmannia roothepatic ischemia-reperfusion injuryoxidative stressferroptosis
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