A Study On The Intervention Mechanism Of Pinocembrin And Gallic Acid Derivatives-rich Fraction Of Penthorum Chinense Pursh In Systemic Lupus Erythematosus

Objective Some studies have reported the urinary protein attenuation capability and the alleviative activity on renal injury in nephrotic syndrome model rat of the total flavonoids fraction in Penthorum chinense Pursh extract.And our previous study also investigated that the pinocembrin and gallic acid derivatives-rich fraction of Penthorum chinense Pursh(PGF)had the lupus model mice protective activity.Therefore,the network pharmacological methods were applicated to predict the molecular mechanism of PGF in this work.In further,the predicted mechanisms were demonstrated with experiments in vivo and in vitro.Methods 1.The experiments with SLE model mice:(1)MRL/lpr lupus mice were engaged in this study,and divided into three groups:model group,prednisone treatment group(PAT group)and the PGF administrated group,and MRL/Mpj mice was control group;(2)Urine protein detection:The urine of each mouse was collected at the 4th and 6th weeks,respectively.And the protein content was detected by a coomassie brilliant blue kit;(3)The blood was retro-orbital collected,and separated into two parts;one was for routine blood tests,the other was for cytokines and anti-nuclear antibodies quantification by FCM method or ELISA kits;(4)At the end of the experiment,the mice were weighted.And the thymus and spleen were collected and weighted to calculate the spleen/thymus index,respectively;(5)The mouse kidney was stained with H&E,PAS and Masson,respectively and morphologically observed under microscope.2.Network pharmacology:(1)The ingredients in PGF were collected from literatures and previous publications.The targets of SLE were collected through GEO,OMIM,PharmGKB databases,et al.(2)The targets of SLE and PGF ingredients were merged to obtain the crucial targets.And the interaction of these targets was presented as PPI network.The GO function and KEGG pathway analysis were engaged in the further study.(3)Based on the KEGG results,the key targets were selected for molecular docking analysis with the selected ingredients,respectively,to verify the molecular-target docking affinity.3.The study with cell assay in vitro:(1)The PGF and PGF-serum were prepared.(2)The cytotoxicity of PGF and PGF-serum on HK-2 cells were determined with MTT assay test using.And the concentration of H2O2 in oxidative stress assay test were also determined with MTT assay in this study.(3)The regulative effect of PGF on the antioxidant response element ARE was evaluated with double fluorophore enzyme reporter assay.(4)The regulative effects of PGF-serum(final concentration:7.5%and 10%)on the translational and transcriptional expression and activation of HIF-1α,TLR4 and NF-κB in HK-2 cells 239T cells,HepG2 cells were determined with Western-blot and real time-PCR method.In compare,the regulative activity of PGF(10,20,40 μg/mL)was also quantified in this study.(5)Additionally,the identical activities of(4)on of PGF-serum(final concentration:7.5%and 10%)on the lymph node and spleen cells,isolated from lupus mice,were determined with the methods.Results 1.The experiments with SLE model mice:In compare with the model group,the spleen/thymus index,the leukocytes and neutrophils in blood from the PGF group and the PAT group were reduced,and the levels of anti-dsDNA antibodies and anti-snRNP/Sm antibodies in the serum were also significantly reduced(P<0.05);the concentrations of IL-12,IFN and TNF-α in the serum were also significantly reduced(P<0.05).The attenuation of histopathological changes of MRL/lpr mice by PAT and PGF treatment were observed,such as the increased inflammatory cell infiltration in the glomeruli and the surrounding tissues,severe hyperplasia of the intra-glomerular thylakoid and basement membrane,and the fibrosis in the intra-glomerular tissues.These results indicated that identity to that of PAT,PGF could reduce the lupus injury of MRL/lpr mice.2.Network pharmacological study:(1)A Total of 20 different active ingredients in PGF were collected.And by screening the targets of components and diseases in the database,143 specific targets of PGF on SLE were obtained.(2)These PGF-SLE targets were studied as following;PPI core network analysis,GO function and KEGG pathway analysis.The GO functional analytic results revealed that the effective mechanism of PGF on SLE might mainly be in the response to hypoxia,immunity and inflammation.And the enrichment of KEGG pathway analysis revealed that cancer related,oxidative stress(HIF-1α),inflammation(NFκB,TLR4)and et.al.pathways were intensively engaged in PGF on SLE treatment.(3)The results of docking analysis showed the good binding properties of specific ingredients,S2 and S3,in PGF on the crucial targets,HIF-1α,NF-κB,and TLR4,of which the binding energies were all lower than 7 kcal/mol.Therefore,it should be verified that the HIF-1α/NF-κB/TLR4 pathway might be the key pathway of PGF on SLE prevention and treatment.3.The study with cell assay in vitro:(1)The cytotoxicity test results showed that:the 2.5%to 15%PGF-Serum showed no significantly cytotoxic to the HK-2 cells;40 μg/mL PGF exhibited a significant toxic effect on HK-2 cells;(2)The double fluorophore reporter assay results showed that PGF treatment could stimulate the transcription of the antioxidant element ARE in 293T cells,and also attenuate the inhibitory effect of H2O2 on ARE.(3)PGF could significantly inhibit the LPS induced overexpression of HIF-1α,TLR4 and NF-κB in 293T and HK-2 cells.(4)PGF could significantly inhibit the LPS stimulated transcriptional expression of HIF-1α,NF-κB and TLR4 after in HK-2 cells.However,this activity was not significant in 293T cells.(5)PGF could also significantly inhibit the transcriptional expression of HIF-1α,NF-κB and TLR4 in spleen cells,which were isolated from MRL/lpr mice,at a low dose;but only TLR4 in lymph node cells was ameliorated with the same in vitro assay.Conclusion 1.PGF can significantly reduce spleen/thymus index,decrease the serum proinflammatory cytokines,reduce urinary protein levels in lupus mice,and attenuate inflammatory infiltration and fibrotic proliferation in glomeruli and surrounding tissues of MRL/lpr mice.2.The PGF can alleviate SLE kidney injury through anti-inflammatory,antioxidant and immune-modulation.3.The PGF can activate the transcription of the antioxidant element ARE and inhibit the transcription and translation of HIF-1α,TLR4 and NF-κB,as well as the phosphorylation of NF-κB.