Study On The Mechanism Of Qinling Solution In Inhibiting Immune Inflammatory Injury Of Renal Tissue In Patients With Hyperuricemia And Analysis Of Its Clinical Efficac

Hyperuricemic nephropathy is a kind of renal disease induced by the metabolism disorder which can lead to hyperuricemia.The hyperuricemia leads to the deposition of urate crystal in reanl tissue which has a mainly side effect on the renal tubule and interstitium and can lead to tubulointerstitial inflammation,kidney calculi,renal fibrosis,chronic renal insufficiency and renal failure.With the development of the research,it is found that hyperuricemia can induce renal inflammatory injury which is an important reason for the occurrence and development of hyperuricemic nephropathy,and inhibition of the renal inflammatory injury is a new target for treatment of hyperuricemic nephropathy.TLR,as a pattern recognition receptor,is a bridge linking innate and adaptive immune responses,as well as the subject of immune and inflammatory responses.TLR4 is the most studied member of the TLR family,through the MyD88 dependent and independent pathway,which can activate the signals such as NF-κB to mediate inflammatory response.It plays an important role in renal inflammatory injury of hyperuricemic nephropathy,and aslo an important entry point to explore the drugs and methods to inhibit the renal inflammatory injury of hyperuricemic nephropathy.Qinlingye,which is created by Professor Meng Fengxian,is a prescription for the treatment of hyperuricemic nephropathy.It can tonify kidney and reduce turbidity,clear heat and detoxify,promot diuresis and disperse blood stasis,and has remarkable clinical curative effects.This paper will discuss my works in two parts:(1)Experimental Researches:in this study,with the starting point of TLR4/NF-κB signal pathway,and on the basis of a combined studies of in vivo and in vitro,we observe the effects of Qinlingye on rat models of hyperuricemic nephropathy and HKC cell which was under stimulating of UA,and to explore its mechanism of inhibiting the renal immune inflammatory injury in hyperuricemic nephropathy;(2)Clinical Research:to observe the effects of QLY on clinical symptom,syndrome and laboratory tests of hyperuricemic nephropathy patiets,and to explore its curative effect and safty.The aim is to provide new ideas and interventions in treatment of hyperuricemic nephropathy.1 Experimental Researches1.1 Vivo ExperimentObjective:To observe the effect of Chinese herbal compound Qinlingye decoction(QLY)on the immune inflammatory factors in serum and renal tissue of rat models with hyperuricemic nephropathy,and to explore its mechanism of inhibiting the renal immune inflammatory injuries in hyperuricemic nephropathy.Methods:Male Sprage Dawley rats were gavaged adenine and feeded yeast for 18 days,and then the sUA and PRO/24h were detected.Models were regarded as the successful models if the sUA and PRO/24h were higher than normal control group(P<0.05).The sucessful models(n=60)were randomly divided into model,positive drug,and high-,medium-,low-dose of QLX,and administrated with distilled water(10ml.kg-1.d-1/i.g),allopurinol(23.33mg.kg-1.d-1/i.g)and QLX(36.4g.kg-1.d-1/i.g,18.2g.kg-1.d-1/i.g and 9.1g.kg-1.d-1/i.g)respectively.Another 6 rats were used as the control group and given distilled water(10ml.kg-1.d-1/i.g).At the end of 6th and 8th week,half of rats in each group were sacrificed and the kidneys and serums were harvested for detecting.RT-PCR was used to detect the mRNA transcription of RANTES、MCP-1、VCAM-1、IL-6 in renal tissue.ELIS A was used to detect the protein expression of RANTES、MCP-1、VCAM-1、IL-6 in serum.Western blot and Immunohistochemistry were used to detect the protein expression of TLR4、NF-κB in renal tissue.Results:①RT-PCR:Compared with the normal control group,level of VC AM-1 mRNA transcription at 6th week and levels of MCP-1、RANTES and IL-6 mRNA transcription at 8th week in the model group were higher(P<0.05,P<0.01);compared with the model group,levels of MCP-1,RANTES and IL-6 mRNA transcription in medium dose group of QLY were lower,levels of MCP-1,RANTES mRNA transcription in low dose group of QLY were lower(P<0.05,P<0.01).②Western blot:Compared with the normal control group,levels of TLR4、NF-κB protein expression at 6th and 8th week in the model group were higher(P<0.01);compared with the model group,levels of TLR4、NF-κB protein expression in three groups of QLY at 6th and in medium-,low-dose groups of QLY at 8th week were lower(P<0.05,P<0.01).③ELISA:Compared with the normal control group,levels of MCP-1,RANTES,VCAM-1 and IL-6 protein expression in the model group at 6th and 8th week were higher(P<0.05,P<0.01);compared with the model group,levels of MCP-1,RANTES,VCAM-1 and IL-6 protein expression in three groups of QLY at 6th week were lower,levels of MCP-1,RANTES,VCAM-1 and IL-6 protein expression in medium-,low-dose groups of QLY at 8th week were lower(P<0.05,P<0.01).④Immunohistochemistry:Compared with the normal control group,level of NF-κB protein expression at 6th and levels of TLR4、NF-κB protein expression at 8th week in the model group were higher(P<0.05,P<0.01);compared with the model group,level of NF-κB protein expression in three groups of QLY at 6th week were lower,level of TLR4 protein expression in medium dose group of QLY and levels of TLR4、NF-κB protein expression in medium-,low-dose groups of QLY at 8th week were lower(P<0.05,P<0.01).Conclusion:QLY may ameliorate the renal immune inflammatory injuries of hyperuricemic nephropathy rats by down-regulating the expression of inflammatory factors via inhibiting the TLR4/NF-κB signal pathway.1.2 Vitro ExperimentObjective:To investigate the effect of Qinglingye extract(QLYE)on TLR4/NF-κB in HKC cells,and to explore its mechanism of inhibiting the renal immune inflammatory injuries induced by uric acid.Methods:HKC cells were cultured,and the model was built by UA-simulated.HKC cells were induced by UA of 26umol/L in model group.While stimulated by UA,the administered groups were intervened by high-,middle-,low dose of QLYE(20,10,5umol/L).After 24,36,48 hours of intervention,the cell media,total RNA and protein were extracted.RT-PCR was used to detect the mRNA transcription of TLR4,NF-κB.Western blot were used to detect the protein expression of TLR4,Iκ-Bα.ELISA was used to detect the protein expression of TNF-α.Results:①RT-PCR:Compared with the control group,the mRNA transcription of TLR4,NF-κB at 36h and NF-κB at 48h in model group were higher(P<0.05);compared with the model group,the mRNA transcription of TLR4 at 36h in QLYE groups were lower(P<0.05);the mRNA transcription of NF-κB at 36h in high-,middle-dose groups and at 48h in high-,low-dose groups were lower(P<0.05,P<0.01).②Western blot:Compared with the control group,the protein expression of TLR4 was higher and Iκ-Bα was lower at 24,36,48h in model group(P<0.05,P<0.01);compared with the model group,the protein expression of TLR4 at 24,36h in high-,middle-dose groups were lower(P<0.05);the protein expression of Iκ-Bα at 24h in low-dose group was higher and at 36,48h in middle-,low-dose groups were higher(P<0.05,P<0.01).③ELISA:Compared with the control group,the protein expression of TNF-α at 24,36,48h in model group were higher(P<0.01);compared with the model group,the protein expression of TNF-α at 24,36,48h in QLYE groups were lower(P<0.01).Conclusion:QLYE may reduce the immune inflammatory reaction of HKC cells induced by UA by inhbiting TLR4/NF-κB signal pathway.It is possibly a important mechanism of inhibiting the renal immune inflammatory of hyperuricemic nephropathy.2 Clinical ResearchObjective:To observe the effects of QLY on change in kidney function,clinical symptom,syndrome and laboratory tests of hyperuricemic nephropathy patiets,and to explore its curative effect and safty.Methods:We enrolled 88 diagnosed hyperuricemic nephropathy patients from March 2015 to February 2017 in Dongfang Hospital,and the patients were randomly assigned in a 1:1 ratio to control group and treatment group.The control group received the allopurinol pills in addition to standard medication,while the treatment group was treated with QLY decoction based on the control group.The treatment time was 3 monthes.The safty was evaluated during the treatment time,and the curative effect was also evaluated after 3 monthes.(1)the primary outcome:change in eGFR category with confirmation based on percent change in eGFR;(2)secondary outcomes:The levels of sUA,sCR,BUN,β2-MG,Hcy and PRO of UN patients before and after three-month treatment were determined.Moreover,the score of signs and symptoms was also examined.Results:We enrolled 88 cases in the early stage,and the 14 of them were shedding.In the end,we actually enrolled 74 cases,of which 35 cases in the control group,39 cases in the treatment group.The graner,age,history of alcohol and disease and disease condition of the two groups were consistent and comparable.After three monthes treatment,all the tagets were examined.(1)the primary outcome:Compared with the baseline,the level of eGFR in two goups were higher(P<0.01),the GFR category in treatment group was lower(P<0.01);compared with the control group,the level of eGFR was higher and the GFR category was lower in in treatment group(P<0.05,P<0.01).The curative effect:The ratio of control group was 17.14%,and the ratio of treatment group was 58.97%.Compared with the control group,the ratio of treatment group was higer(P<0.01).(2)secondary outcomes:①Laboratory tests:Compared with the baseline,the level of sUA in two goups were lower(P<0.01),the levels of sCR,Hcy and PRO in treatment group were lower(P<0.05,P<0.01);compared with the control group,the levels of sUA,sCR,Hcy and PRO in treatment group were lower(P<0.05,P<0.01).②The score of signs and symptoms:Compared with the baseline,the scores of signs and symptoms in two groups were lower(P<0.05,P<0.01);compared with the control group,the score of signs and symptoms in treatment group was lower(P<0.05).There were no adverse reactions and abnormal safty indexes in two groups.Conclusion:Combination treatment of allopurinol pills and QLY decoction on the basic treatment not only improve kidney function,the clinical symptom and syndrome,but also reduce the levels of sUA,sCR,PRO and Hcy.Based on the research,we find that the combination treatment can treat hyperuricemic nephropathy effectively and protect the renal function.