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OBJECTIVE: To investigate the protective effects and mechanism of extracts of Rehmanniae against atherosclerosis by “IL-34”-Rho/Rock pathway in ApoE-/-mice. METHODS: ①Animal experiment 40 ApoE-/- mice were randomly divided into CTL group, Model group, Reh-L group (5 μg·g-1) and Ren-H group (10 μg·g-1), which were induced by low-density lipoprotein (LDL) for AS models. The serum levels of interleukin-34 (IL-34), LDL, mouse monocyte chemotactic protein 1 (MCP-1), mouse macrophage inflammatory protein 2(MIP-2) and endothelin-1 (ET-1) were detected by ELISA. AS index (AI) was calculated according to the serum lipids levels. ②Macrophage experiment RAW264.7 macrophages were cultured in vitro and divided into con group, ox-LDL group, IL-34 group and Reh group. IL-34-indeced foam cells formation by oil red O staining. The cholesterol ester levels and cholesterol efflux of RAW264.7 macrophages were detected. The immunofluorescence, qRT-PCR, Western blot analysis were used to detect RhoA and Rock1 expression. RESULTS: ①Animal experiment the serum levels of TC, TG, LDL-C, ET-1, IL-34, MCP-1, MIP-2 and AI, atherosclerotic lesion areas of the thoracic aorta of mice in Reh-H group were lower than Model group and Reh-L group, but with higher HDL-C level (P<0.05). Compared with the CTL group, the RhoA and Rock1 levels of aorta tissues in Model group were significantly increased by qRT-PCR and Western blot (P<0.05). But the RhoA and Rock1 levels of aorta tissues in Reh-H group were lower than Model group or Reh-L group (P<0.05). ②Macrophage experiment the cholesterol ester level and cholesterol efflux, RhoA and Rock1 expressions of RAW264.7 macrophages induced by ox-LDL and IL-34 were more than con group and ox-LDL group (P<0.05). Compared with IL-34 group, the cholesterol ester level and cholesterol efflux, RhoA and Rock1 expressions of RAW264.7 macrophages in Reh group were significantly decreased (P<0.05). CONCLUSION: The extracts of Rehmanniae can protectagainst atherosclerosis in ApoE-/- AS mice models induced by LDL through inhibiting “IL-34”-Rho/Rock pathway.
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Objective:To investigate the effect of IL-34 on the phenotype of monocyte derived dendritic cells in RA,and to speculate the role of IL-34 in the differentiation of myeloid dendritic cells.Methods:The peripheral blood of RA patients was collected to harvest PBMC by Ficoll density gradient centrifugation and cultured for 4h.Adherent cells were stimulated with GM-CSF+IL-4,IL-4, IL-4+IL-34 for 3 days,and then the expression of CD83,CD86 and CD14 was tested by flow cytometry.In addition,the cells stimulated by GM-CSF and IL-4 were added by TNF-α with or without IL-34 for another four days.The expression of CD83,CD86 and/or CD14 was detected by flow cytometry.Results:(1)The expression of CD83 and CD86 on immature DC induced by IL-34+IL-4 was upregulated compared with IL-4 alone(P<0.01),but no difference of the CD14 levels between the two groups(P>0.05).The levels of CD86 and CD14 induced by IL-34+IL-4 were slightly decreased compared with GM-CSF+IL-4 stimulation(P<0.05),but no difference of CD83 expression between the two groups(P>0.05).(2)The expression of CD83 and CD86 stimulated by GM-CSF+IL-4+IL-34 was lower than the GM-CSF+IL-4+TNF-α group(P<0.05),but no difference compared with GM-CSF+IL-4 group(P>0.05). (3)The CD83 expression induced by GM-CSF+IL-4+TNF-α+IL-34 was lower than GM-CSF+IL-4+TNF-α group(P<0.05),but there was no difference of CD86 and CD14 expression between the two groups(P>0.05).Conclusion:IL-34 played roles in the process of immature DC differentiation,but the effect was slightly weaker than that of GM-CSF.IL-34 did not effect the phenotype change of mature DC,but involved in the maintainence of immature DC.
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Objective To investigate the expression of interleukin-34 (IL-34) after corneal transplantation in rats and its mechanisms of action in immune rejection.Methods SD rats were used as donors and Wistar rats as the recipients to establish corneal transplantation experimental models.Together 60 Wistar rats were randomly divided into 3 groups according to the random number table methods,and rats in B group was transplantated with autologous cornea,while C and D groups received allogeneic corneal transplantation.Another 10 rats were collected as normal controls (A group).After B,C group was treated with postoperative Tarivid eye drops,D group was treated with TobraDex eye drops postoperatively.The survival rate of corneal grafts and survival analysis were evaluated in the 10 rats from the 4 groups,and then the corneal grafts were taken from the rest of rats at the fourteenth day after the operation for hstopathological,immunohistochemical and RT-PCR examinations.Results Survival analysis showed that immune rejection did not occur in A and B group,and the mean survival time (MST) was (26.00 ± 0.97) days in D group,which was much higher than that in group C[(10 ± 1.55) days] (P ＜0.001).HE staining showed that there was obvious inflammatory cell infiltration and neovascularization in the corneal tissue of C group,and there were only a few inflammatory cells and neovascularization in D group.Immunohistochemistry showed that IL-34 protein expression in C group (0.089 4 ± 0.005 6) was significantly higher than that of A group (0.037 7 ± 0.002 3),B group (0.068 4 ±0.004 4) and group D (0.044 5 ± 0.004 5) (F =145.21,P ＜ 0.01),and its expression was mainly located in the epithelial and stromal layer.RT-PCR showed that the expression levels of IL-34,IL-1β,IL-17A,TNF-α mRNA in corneal tissue of C group were significantly higher than those in A,B and D group (all P ＜ 0.05).Conclusion IL-34 may involve in the rejection after corneal transplantation in rats,and TobraDex eye drops can delay the rejection by inhibiting the expression of IL-34 and related signaling pathways.
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@#[Abstract] Objective： ：To study the effects of IL-34 over-expression on malignant biological behavior of acute monocytic leukemia (AMoL) cells. Methods: The lentiviral vector pCDH-GFP for over-expressing IL-34 was constructed and infected into AMoL cell lines (THP1 and MOLM-13). Then its effects on proliferation, colony forming and cell cycle as well as apoptosis were tested by the MTS, colony formation assay and Annexin-V/PI staining, respectively. The cell differentiation phenotypes were assessed by fflow cytometry. Nude mice xenograft model was established to observe the tumor size and mass as well as the macrophages recruitment. Results: qPCR analysis showed that the expression of IL-34 mRNAin THP1-IL-34 and MOLM-13-IL-34 cells was nearly 4 000 and 3 000 folds higher than their respective control cells (all P<0.01), indicating that AMoL cell lines over-expressing IL-34 were successfully established. In vitro study showed that over-expression of IL-34 in AMoL cell lines promoted their proliferation potential(72 h:[0.738±0.003] vs [0.646±0.008]; [0.290±0.004] vs [0.247±0.004]; all P<0.01) and colony formation ([127.00 ± 3.37] vs [86.00±4.08]; [160.70±4.70] vs [116.70±3.93]; all P<0.01), whereas had little effect on apoptosis (all P>0.05). Over-expression of IL-34 promotedAMoLcell differentiation towards monocyte-macrophage lineage as the expressions of the monocyte-macrophage markers, CD11b and CD14, were increased whereas the expression of immature marker, CD71, was decreased in AMoL cell lines over-expressing IL-34(all P<0.05). Nude mice xenograft model showed that IL-34 over-expression stimulated macrophage recruitment in tumor tissues (P<0.01). Conclusion: Over-expression of IL-34 in human AMoL cell lines promotes their proliferation, colony forming potential and differentiation towards monocyte-macrophage lineage. Furthermore, IL-34 participates in the process of macrophages recruitment in vivo.
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Objective:To investigate levels of serum IL-34 in SLE patients,and correlation between concentrations of serum IL-34 and other established serum markers and disease activity indexes.Methods: In all,78 SLE patients and 53 healthy controls were enrolled in the research.Enzyme-linked immunosorbent assay(ELISA) was employed to measuring the concentrations of serological IL-34.Then,serum IL-34 levels between SLE group and healthy controls were analyzed by Mann-Whitney U test.Meanwhile,the correlation between the serum IL-34 levels and disease activity indexes and other established serum markers were assessed.Serum IL-34 levels were significantly higher in SLE patients compared to healthy controls[(Median,128.9 pg/ml) vs (Median,52.4 pg/ml),P<0.001].Results: Their levels were remarkably associated with accumulation of the clinical features of SLE.Additionally,IL-34 titers were positively correlated with the SLE disease activity indexes,anti-double stranded DNA antibody(anti-dsDNA)titers and C-reactive protein(CRP)levels,but inversely with C3 levels.Conclusion: Serum IL-34 could be a candidate biomarker for SLE as the elevated serum levels in treatment-naive SLE patients and its association with SLE disease activity.
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To construct recombinant eukaryotic expression plasmid vector of human IL-34 gene, and to study the effects of IL-34 expressed by human bone marrow-derived mesenchymal stem cells (hBM-MSCs) on THP-1 cells. Full-length IL-34 encoding sequence was amplified by PCR. And this fragment was cloned into the plasmid pIRES2-EGFP. Western blotting and ELISA were used to analyze the expression of IL-34 in hBM-MSCs. THP-1 cells were cultured with hBM-MSCs medium containing IL-34 protein. Real-time PCR detected the effects of IL-34 on the expression of IL-10 and TNFα in THP-1 cells. Restrictive enzyme analysis and sequencing demonstrated that IL-34 eukaryotic expression vector was successfully constructed. IL-34 protein expressed by hBM-MSCs could promote IL-10 and TNFα expression in THP-1 cells. Those results show that IL-34 expressed by hBM-MSCs has regulating effect on THP-1 cells.