The Role Of 12(S)-HETE In The Expression Of Interleukin-6 In Diabetic Glomeruli

Background:As one of the most important complications of diabetes mellitus(DM),diabetic nephropathy(DN)is one of the most important causes of end-stage renal failure worldwide,and has become an increasingly serious problem in human health.Therefore,it is of great significance for the health of the whole human being to clarify the pathogenesis of DN,study how to delay the progress of DN and seek effective treatment methods actively as soon as possible.In recent years,more attention has been paid to the pathogenesis of DN in the cell and molecular level,which makes people realize that inflammation can play an important role in the pathogenesis of DN.Among many inflammatory factors,the role of interleukin-6(IL-6)has been getting attention increasingly.At present,it has been found that overexpression of IL-6 in DN is associated with glomerular hypertrophy and urinary protein excretion,and can accelerate the progression of DN through a variety of mechanisms.However,there are no reliable and detailed reports on the factors affecting the expression of IL-6 in DN.The study showed that in the cell or animal model of hyperglycemia,the level of12(S)-hydroxyeicosatetraenoic acid [12(S)–HETE],which was the production of12-lipoxygenase(12-LO),was increased significantly,and 12(S)-HETE can interact with angiotensin?(Ang?)and transforming growth factor-?(TGF?)and then affect the progress of DN together.Associating with the role of IL-6 in DN that we have already recognized,we assume that 12(S)-HETE may also accelerate the progression of DN by up regulating the expression of IL-6 in type 1 or type 2 DM.It has been found that p38 MAPK that belongs to mitogen-activated protein kinase(MAPK)acts as the junction of the cell signal transduction pathway,which can be activatedby a variety of factors in DN.In recent years,many studies have shown that p38 MAPK plays an important role in the early stage of DN,also in the development phase.In addition,p38 MAPK can also plays an important role in the pathogenesis of insulin resistance(IR)by regulating multiple transcription factor,protein and enzyme activity.As a result,a lot of attention has been paid to the study of the role of p38 MAPK in the pathogenesis of DN.However,there is no relevant report on whether p38 MAPK signaling pathway can play an important role as an intermediate link between 12(S)-HETE and IL-6.In recent years,several evidences have indicated that gene polymorphism plays an important role in determining the susceptibility of diabetic nephropathy.Since Fishman et al found that the polymorphism of IL-6 gene in-174G>C can affect the transcription and serum level of IL-6 gene,IL-6 gene polymorphism has become a hot research topic.Since the IL-6 gene-174G>C and-634C>G have important functions,they are the 2 most studied single nucleotide polymorphisms.However,the results of different studies on the relationship between these two polymorphisms and DN susceptibility are not identical,so the question of whether the IL-6 gene polymorphism affects the susceptibility of DN has not been solved.In conclusion,this paper puts forward two suggestions: first,12(S)-HETE can up regulate the expression of IL-6 by activating p38 MAPK pathway,and then accelerate the progress of DN;Inhibiting 12(S)-HETE-p38 MAPK way can delay the development of DN.Second,IL-6 gene polymorphism is associated with DN susceptibility.Methods:(1)12(S)-HETE with the concentration of 10-7mol/L was used to stimulate rat mesangial cells(RMC)directly,and then the cells were collected after stimulation of 0h,1h,2h,4h,6h,24 h,and the expression of IL-6 was measured.(2)The primary cultured mice mesangial cells(MMC)were divided into two groups:the wild MMC group(WT)and the 12-LO gene knockout MMC group(LOKO),and the expression of IL-6 was measured.(3)The MMC of the transgenic mice were divided into two groups: the 12-LO overexpression group(pc DNA/12-LO)and the 12-LO normal expression group(pc DNA),and the expression of IL-6 was measured.(4)Primary cultured RMC were divided into 3 groups: normal control group,12(S)-HETE treatment group and 12(S)-HETE+SB203580 treatment group(SB203580 as an inhibitor of p38 MAPK,the concentration of 1 ?mol/L,used 30 minutes before giving 12(S)-HETE).The expression of IL-6 and the levels of p-p38 MAPK in three groups was measured after 24 h.(5)Rats were divided into 2 groups: 12(S)-HETE treatment group(pumping12(S)-HETE by osmotic mini-pumps,1 mg·kg-1·d-1)and control group,the rats were sacrificed 7 days later,the levels of IL-6,p-p38 MAPK,p-ERK and p-JNK in glomeruli were detected.(6)The model of type 1 DM was induced by intraperitoneal injection of large dose of streptozotocin(STZ).The DM mice were divided into 4 groups: the wild group(WT),the12-LO gene knockout group(LOKO),the wild DM group(WT+STZ)and the 12-LO gene knockout DM group(LOKO+STZ).Blood glucose,body weight and 24 h urine albumin were detected before the end of the experiment.After the rats were sacrificed,the right kidney was weighed,the glomeruli were separated and the glomerlular IL-6 levels were detected.(7)High-fat diet combined with low dose STZ intraperitoneal injection were used to induce type 2 DM model,and after the success of the model,DM rats were randomly divided into 2 groups: type 2 DM group and type 2 DM+ cinnamyl-3,4-dihydroxy-?-cynanocinnamate(CDC)group(CDC as the inhibitor of 12-LO,subcutaneous injection,8mg·kg-1·d-1,3/week),with standard diet fed rats as control group.After 8 weeks,blood glucose,body weight and 24 h urine albumin were measured.After the rats were sacrificed,the right kidney was weighed,and the glomeruli were separated.The pathological structure of the kidney was observed and the levels of IL-6 and p-p38 MAPK in glomeruli were detected.(8)Meta analysis was used to assess the relationship between IL-6 gene polymorphism and DN susceptibility by calculating pooled odds ratio and 95% confidence intervals.The patients were divided into 3 groups(large albuminuria group,trace albuminuira groupand normal albuminuria group)according to urinary albumin excretion rate for subgroup analysis.Glomeruli were isolated with a sieving method,renal slices were stained with periodic acid schiff(PAS),urine albumin and levels of IL-6 in glomeruli were detected by enzyme-linked immunosorbent assay(ELISA),p-p38 MAPK,p-ERK and p-JNK levels were detected by Western blot,the expression of IL-6 in mesangial cells and glomeruli was detected by reverse transcription-polymerase chain reaction(RT-PCR).Results:(1)After 1 hour of stimulation with 12(S)-HETE,the expression of IL-6 in RMC was increased significantly(P<0.01),and it could be maintained for up to 24 hours.(2)Compared with WT control group,the expression of IL-6 in MMC in 12-LO gene knockout group was reduced significantly(P<0.01).(3)Compared with the control group,the expression of IL-6 in MMC in 12-LO overexpression group was significantly higher(P<0.01).(4)Compared with the control group,the p38 MAPK activity and the expression of IL-6in RMC in the 12(S)-HETE treatment group was increased significantly(P<0.01).Compared with the 12(S)-HETE treatment group,the inhibition of p38 MAPK by SB203580 could decrease the p38 MAPK activity and the expression of IL-6 in RMC in the 12(S)-HETE +SB203580 group significantly(P<0.05).(5)Compared with the control group,the expression of IL-6 in glomeruli in 12(S)-HETE treatment group was increased significantly(P<0.01)(6)Compared with the control group,12(S)-HETE treatment group had no significant changes in the levels of p-ERK and p-JNK in glomeruli,but the levels of p-p38 MAPK in glomeruli in 12(S)-HETE treatment group were increased significantly(P<0.01).(7)Compared with WT group and LOKO group,the blood glucose and urine albumin of mice were increased significantly in WT+STZ group and LOKO+STZ group(P<0.01).Compared with WT+STZ group,LOKO+STZ group had no significant changes in blood glucose or urine albumin.(8)Compared with WT group and LOKO group,the kidney weight / body weightratio and the expression of IL-6 were increased significantly in WT+STZ group(P<0.01).Compared with WT group and LOKO group,the kidney weight / body weight ratio and the expression of IL-6 were increased significantly in LOKO+STZ group(P<0.05).Compared with WT+STZ group,the kidney weight / body weight ratio and the expression of IL-6 were decreased significantly in LOKO+STZ group(P<0.05).(9)Compared with the control group,the blood glucose,kidney weight / body weight ratio and 24 h urine albumin were increased significantly in the type 2 DM group(P<0.01).Compared with type 2 DM group,after 8 weeks of CDC treatment,there was no significant change in blood glucose in the type 2 DM+CDC group,but the renal weight / body weight ratio and urine albumin decreased significantly(P<0.05).(10)Compared with the control group,the glomeruli volume in type 2 DM group was increased significantly(P<0.01),as well as the proliferation of mesangial matrix.After 8weeks of treatment with CDC,compared with type 2 DM group,the glomeruli volume in type 2 DM+CDC group was reduced significantly(P<0.05),and the proliferation of mesangial matrix was relieved.(11)Compared with the control group,the levels of IL-6 and p-p38 MAPK in glomeruli of rats with type 2 DM were increased significantly(P<0.01).After 8 weeks of CDC treatment,the levels of IL-6 and p-p38 MAPK in glomeruli in type 2 DM+CDC group were decreased significantly compared with that in type 2 DM group(P<0.05).(12)There was no significant relationship between the-174G>C polymorphism of IL-6 gene and the DN susceptibility,while the-634C>G polymorphism of IL-6 gene could increase the DN susceptibility.The results of subgroup analysis were consistent with the results of the whole model.Conclusions:(1)12(S)-HETE can increase the expression of IL-6 in the glomeruli of DN animals;inhibition of 12(S)-HETE can reduce the expression of IL-6 in the glomeruli of DN animals,and can inhibit glomeruli hypertrophy and delay the progression of DN.(2)12(S)-HETE induces the expression of IL-6 in glomeruli through the p38 MAPK pathway,and inhibition of p38 MAPK can reduce the expression of IL-6.(3)The-634C>G polymorphism of IL-6 gene could increase the DN susceptibility,but there was no significant correlation between-174 gene G>C polymorphism of IL-6 and DN susceptibility.