| Objective:Ischemia-reperfusion injury(IRI) is refers to the tissues or organsrestoration of blood flow in the ischemic basis, leading to the aggravated damagephenomenons of cell metabolism and structure. Acute kidney injury (AKI) is a commonclinical disease, its prevalence is rising year by year, AKI is defined as increased serumcreatinine(Scr)≥26.5umol/l within48h; or serum creatinine increase(BUN)≥1.5timesthe baseline value; or the last6h urine output <0.5ml/kg.h. Its pathogenesis is not yetfully understood. Renal IRI is one of the leading causes of ischemic AKI.Research shows that the immune inflammatory reaction is one of the importantmechanisms of IRI, in which inflammatory receptor plays an important role.Toll-likereceptors(TLRs) is a type I transmembrane protein receptor discovered in recent years.First,it can identify and bind the type of pathogen-associated molecular patterns ordamage-associated molecular patterns.Second,it can trigger a series of intracellularsignal transduction, resulting in the release of inflammatory mediators and eventuallyleading to the occurrence of immune inflammatory reaction.There is evidence that TLRs is involved in the cause of immune inflammatoryresponses triggered by ischemia.Also TLRs is the key sensor of tissue damage. A largenumber of experimental data in support of TLRs act as a new target for inflammatoryand autoimmune diseases.Drug research on blocking or inhibiting TLRs signalingpathway node has become a hotspot in the field of medicine. Currently, however, thereis no effective method of western medicine on prevention and treatment of renal IRI.Traditional Chinese medicine for thousands of years has played an indispensable role indifferentiation therapy.Traditional Chinese medicine formulas studies are now attractingmore and more attention around the world.In this paper, through the establishment of a rat model of renal IRI,we explored therenoprotective effect on AKI induced by IRI between low-dose and high-dose compound traditional Chinese medicine-Fufang Shenhua Tablet(SHT).At the same time,protein and mRNA expression of toll-like receptor2(TLR2) and toll-like receptor4(TLR4) was observed within1week postoperative IRI,as well as myeloiddifferentiation factor88(MyD88) and inflammatory cytokines associated to TLRs signaltransduction pathway.From the perspective of TLRs signal transduction pathway,toreveal the renoprotective effect and its possible mechanism of SHT on renal IRI in rats.Methods:(1)To established the renal IRI model through occlusion bilateral renal pediclein Wistar rats by the use of non-invasive arterial clip.Depending on the time of renalischemia, rats were randomly divided into sham operation group (Sham),20min group,30min,40min group and50min group. Scr, blood urea nitrogen (BUN) were measuredby automatic biochemical analyzer after ischemia-reperfusion24h.Renal tissue wasconventional processed by ethanol dehydration,paraffin embedding to4μm tissuesections.Damage of renal tubules was observed under optical microscope after PASstaining to compare the effects of different ischemic time on the above renal indexes. Atthe same time,the survival rate of the renal ischemia50min group was postoperativeobserved for one week.(2)After the ideal renal ischemia time was determined,Wistar rats were randomlydivided into two groups, one group of unilateral nephrectomy, unilateral renal pedicleclamping,another group underwent bilateral renal pedicle clamping.Scr and BUN aswell as one week postoperative survival rate of rats were observed after reperfusion for1d,3d,5d,7d.The survival curve was plotted.Screening out the ideal surgical mode andthe renal pedicle clamping time required for rat IRI model.(3) The rats were randomly divided into four groups: sham operation group (Sham),model group (Model), low-dose Fufang Shenhua Tablet group (SHT-L) and high-doseFufang Shenhua Tablet group (SHT-H).Gavage given drugs one time a day to establishthe renal IRI model with bilateral renal pedicle clamping40min method seven dayslater.Vital signs of rats was observed postoperative24h and72h.Scr and BUN weremeasured by automatic biochemical analyzer;Renal tubular injury of the rat kidney wasobserved under optical microscope and was rated the degree of renal tubular injury with computer microscopic image acquisition and analysis system to evaluate and assess therenoprotective effect of various drugs.(4) The rats were randomly divided into five groups: Sham group, Model group,Astragaloside group,SHT-L group and SHT-H group.Various drugs were given bygavage once daily,establishing the renal IRI model with the right nephrectomy, the leftrenal pedicle clamping40min method seven days later.Rats vital signs was observedpostoperative24h and72h.Using automatic biochemical analyzer to measure BUN andScr,and observation under the microscope the degree of tubular injury of the ratkidney;Using microscopic image acquisition and analysis system to score the degree ofinjury;Using JEM-1400electron microscope to observe the ultrastructural changes ofrenal tubular epithelial cells,on which quantitative analysis was done.(5) The rats were randomly divided into five groups: Sham group, Model group,Astragaloside group,SHT-L group and SHT-H group.Various drugs were gavage givenseven days, then the renal ischemia-reperfusion rat model was established. Toll-likereceptors2(TLR2),Toll-like receptors4(TLR4),myeloid differentiation factor88(MyD88),interleukin6(IL-6),interleukin12(IL-12) protein and mRNA expression aswell as protein expression of interleukin8(IL-8) and IFN-gamma (IFN-γ) on the renaltissue were detected by the means of immunohistochemical ABC method (IHC-P) andWestern blot (WB) and real-time quantitative nucleic acid amplified detection system(qPCR) postoperative1d,3d,5d,7d.Results:(1) The success rate of experimental operation model was97.6%, comparedwith Sham group,30min group,40min group and50min group,BUN and Scr and renalhistopathological scores were all increased1d after reperfusion.Compared with theSham group, the differences were statistically significant(P<0.05), especially in thegroups of40min and50min(P<0.01); however, there was no significant differencebetween20min group and Sham group(P>0.05). Comparison Scr and survival ratebetween Unilateral nephrectomy with renal pedicle clamping group and bilateral renalpedicle clamping group for one week, there was no significant difference between thetwo groups(P>0.05). The survival rate decreased significantly in50min group to50%. (2) Comparison between Sham group and Model group postoperative24h and72h,renal function and renal pathological damage in rats were significantly abnormal(P<0.01). Renal function and renal pathological damage in SHT-L group were reducedsignificantly(P<0.05),and which were reduced more significantly in SHT-Hgroup(P<0.01) compared with the Model group postoperative24h and72h.Renalfunction and renal pathological damage in rats of SHT-H group was lower than that ofin SHT-L group (P<0.05).(3)Both renal function and pathological damage were reached the peak after IRI24h detected by renal light microscopy and electron microscopy.The above indicatorsare partial restoration after IRI72h.The quality of rats life,Renal function and renalpathological lesion were improved significantly in SHT-L group(P<0.05).And whichwere improved more significantly in SHT-H group(P<0.01) compared with the Modelgroup postoperative24h and72h.These indicators were similar between Astragalosidegroup and SHT-L group (P>0.05), while the above parameters in SHT-H group werebetter than that of in Astragaloside group and SHT-L group (P <0.05).(4)Compared with Model group,both TLR2and TLR4protein and mRNAexpression in Astragaloside group and in SHT-L group were decreased postoperative1d,3d,5d,7d(P<0.05),which were decreased even more significantly in SHT-H group(P<0.01). Both TLR2and TLR4protein and mRNA expression in various groupsreached the peak after IRI5d.Compared with Astragaloside group and SHT-Lgroup,TLR2and TLR4protein and mRNA expression in SHT-H group were decreasedsignificantly(P<0.05) postoperative1d,3d,5d and7d.While TLR2and TLR4protein andmRNA expression had no significant difference between Astragaloside group andSHT-L group (P>0.05).Compared with Model group,IL-6,IL-12and MyD88proteinand mRNA expression in Astragaloside group,SHT-L group and SHT-H group weredecreased significantly postoperative1d,3d,5d,7d (P<0.05). IL-8and TNF-γ proteinexpression in various groups reached the peak one day after IRI.IL-6and IL-12proteinand mRNA expression in various groups reached the peak three days after IRI.MyD88protein and mRNA expression in various groups reached the peak five days after IRI.Compared with Astragaloside group and SHT-L group,MyD88protein and mRNAexpression as well as IL-8and TNF-γ protein expression were decreased significantlypostoperative1d,3d,5d,7d in SHT-H group(P<0.05).While MyD88protein and mRNAexpression as well as IL-8and TNF-γ protein expression had no significant differencebetween Astragaloside group and SHT-L group (P>0.05).Conclusion:(1) Stable renal IRI model in rats can be built on renal pedicleclamping by the means of non-invasive artery clip.Ideal ischemic time to build on renalmodel was40min in rat. Both unilateral nephrectomy with unilateral renal pedicleclamping method and bilateral renal pedicle clamping method were feasible.The modelwas satisfactory and its postoperative survival rate was high.(2) Both Chinese herbal compound (SHT) and single herb (Astragaloside) cansignificantly improved the quality life of IRI rats, protected their renal function andalleviated their pathological lesion of renal tissue,resulting in a good protective effect onthe kidney of IRI rats.Similar efficacy can be seen between low-dose SHT andAstragaloside, whereas the efficacy of high-dose SHT showed a good dose effect andwas obviously superior to Astragaloside.(3)SHT can inhibit immune inflammatory receptors-TLR2and TLR4protein andmRNA expression in rats.Inhibition of TLR2and TLR4protein and mRNA expressionin SHT-H group was superior to that of in Astragaloside group and SHT-L group,suggesting a role for SHT with good dose effect on the inflammatory immunereceptor-TLR2and TLR4.(4)SHT may possibly through the regulation of MyD88-dependent TLRs signaltransduction pathways to inhibit the release of inflammatory cytokines,and to relieveand partly to repair IRI-induecd AKI in rats with a good dose-dependent effect.Thisstudy demonstrated that TLRs signal pathway mechanism maybe involed in the SHTprevention and treatment of ischemic AKI. |
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