Research Of Monocytes TLR4and VDR Expression In Type2Diabetes Mellitus And Diabetic Nephropathy Uremia Adjustment Inflammatory Response And Intervention Mechanism Of1,25-(OH)₂D3

AimThe differential expression of CD14⁺CD16⁺and TLR4expression inmonocytes and related cell factor levels among T2DM and DN uremiapatients.MethodsTwenty-eight uremic patients without DN, thirty DN patients withuremia, twenty-eight T2DM patients, and twenty healthy volunteers wereenrolled for the determination of CD14⁺CD16⁺fluorescence intensity andTLR4expression on monocytes by using peripheral blood flow cytometry.Serum C-reactive protein (CRP) level was determined by using theimmunoturbidimetry. Serum endotoxin level was determined byquantitative colorimetric limulus test. Concentrations of IL-6and monocytechemoattractant protein-1(MCP-1) in supernatants were assessed by ELISA.ResultsCompared to normal control, T2DM patients and DN uremic patientshad a significantly higher CD14+CD16+fluorescence intensity, TLR4expression, and serum CRP level, whilst these biomarkers were moreupregulated in DN uremic patients than in T2DM patients.But thosebiomarkers were significantly downregulated in uremic patients withoutDN(P <0.01). Compared to uremic patients without DN, DN uremicpatients CD14+CD16+monocyte expression and serum CRP level wereslightly lower, but there were no significant differences (P>0.05).Concentrations of IL-6and MCP-1in supernatants were increasedgradually.And DN uremic patients> uremic patients without DN> T2DMpatients> normal control. Peripheral blood CD14⁺CD16⁺monocyteexpression,serum CRP level were increased and in a positive correlationwith supernatants IL-6and MCP-1concentrations.And the endotoxin(ET)levels were more upregulated in T2DM patients and uremic patientscompared to normal control, especially uremic patients had a significantlyhigher ET level(P <0.01).ConclusionThese findings suggest that the immune disturbance inproinflammatory CD14⁺CD16⁺monocytes occurs at the stage of T2DM.Such immunological dysfunction may be mediated the occurrence and progression of T2DM and DN uremia. The monocytes dysfunction may bealso result in the occurrence of uremic patients without DN.However, DNuremic patients peripheral blood inflammation factors level were increasedsignificantly than in uremic patients without DN patients.It may be one ofthe reasons, which DN becomes the first one cause of end-stage renal failure(ESRF). To explore the effects of1,25-(OH)₂D3and lipopolysaccharide (LPS)plus human recombinant interleukin-15(IL-15) on expression of vitamin Dreceptor(VDR), Toll-like receptors4(TLR4),TLR9, STAT5, NF-κBsignaling pathway and cytoskeletal rearrangement in human monocytesincubated with sera from type2diabetes (T2DM)patients and diabeticnephropathy(DN) patients with uremia. To further investigate its possiblemechanism of1,25-(OH)₂D3in T2DM and DN uremia inflammatoryimmune response.MethodsPeripheral sera were isolated from healthy volunteers (control group),T2DM patients and DN uremic non-dialysis patients. After incubation with or without1,25(OH)2D3, THP-1monocytes were treated with LPS plusIL-15prior to the collection of cells and supernatants.VDR, TLR4, TLR9and IL-15mRNA transcription was examined by Real-time PCR, whilstTHP-1monocytic TLR4, VDR, NF-κBP65, IκB, STAT5and p-STAT5expressions were investigated by Western blotting. Concentrations of IL-6and monocyte chemoattractant protein-1(MCP-1) in supernatants wereassessed by ELISA.Immunofluorescence and a laser confocal microscopywas used to examine the expression of VDR and cytoskeletal proteins. Todetect the effect of AG490on LPS, IL-15,1,25-(OH)₂D3and sera mediatedTHP-1monocytic STAT5phosphorylate level.Results1. The THP-1cells proliferation was promoted which incubated by the5%concentration serum of uremic patients with diabetic nephropathy invitro, and the most significant effect was12h group than the others, whichmechanism may be related to TLR4.2. Compared to the normal control, LPS and IL-15down-regulatemonocytic VDR and IκB expression in T2DM patients and DN uremicpatients, whilst with cytoskeletal rearrangement,they up-regulate TLR4,p-STAT5, NF-κB P65protein expression as well as IL-6and MCP-1activityand TLR4, IL-15mRNA levels. However, there were no significantdifferences in TLR9mRNA and STAT5protein levels among the threegroups (P>0.05). Such effects could be in part blocked by1,25-(OH)₂D3. The aforementioned effects were improved following the pretreatment with1,25-(OH)₂D3. And there were no significant differences in VDR,TLR4mRNA and VDR,NF-κB P65,IκB and p-STAT5protein expression andrelated inflammation factors level of1,25-(OH)₂D3-treated THP-1monocytes on three groups (P>0.05). The cellular shape and the cytoskeletalproteins distribution were normal which on THP-1monocytes.3. Prior to the pretreatment with AG490,compared to the LPS+IL-15group, VD3group,AG490+LPS+IL-15group and AG490+VD3group had asignificantly down-regulate THP-1monocytic STAT5phosphorylate level(P<0.01).And AG490+VD3group was the most remarkable.ConclusionThe above results suggest that the anti-inflammatory mechanism of1,25-(OH)₂D3may be related to TLR4,NF-κB P65,cytoskeletal proteins,VDR and STAT5signaling pathway. To validate the interaction between VDR and p-STAT5. And to furtherelaborate the potential mechanisms of immunoregulatory and anti-inflammatory effects of1,25-(OH)₂D3on T2DM and diabeticnephropathy(DN)patients with uremia.MethodsThe experiment divides into three groups(1)control group(2)LPSand IL-15-treated group(3)1,25-(OH)₂D3pretreated group. VDR andp-STAT5were co-localized by using immunofluorescence. As theimmunofluorescence experiment validated the possible intranuclearinteraction of VDR with p-STAT5, the immuno-coprecipitation and westernblotting assay of VDR was performed to further validate the interaction ofVDR with p-STAT5.ResultsCompared to the normal control, LPS+IL-15-treated group and1,25-(OH)₂D3pretreated group p-STAT5was expressed in the nuclei, VDRinteracted with p-STAT5, and1,25-(OH)₂D3pretreated group had asignificantly increasing.ConclusionOur results showed that VDR and p-STAT5possible to have interactionin the THP-1monocyte. The anti-inflammatory role of1,25-(OH)₂D3ininflammatory might be associated with STAT5-VDR cross-talk. Thiscross-talk might further explain why the vitamin D insufficient patient easyinfection,and the anti-inflammatory role of1,25-(OH)₂D3in inflammatoryand immune response of T2DM and DN might be associated with STAT5-VDR cross-talk. Therefore, the appropriate supplementation of1,25-(OH)₂D3might be prevention and protection for vitamine-D-deficientsubjects(such as DM and DN patients).