Study Of QZSZ Particle On Thioredoxin System And Urinary Micrornas-192,29c Expression In Patients With Refractory Nephrotic Syndrome

Primary nephrotic syndrome (PNS) is characterized by a massive proteinuria, hypoalbuminemia, edema, etc of a kind of clinical common kidney disease, after standard therapy of the hormone to treat disease, can’t effectively relieve and accur hormone resistance, dependency or invalid or frequent recurrence, which in turn become refractory nephrotic syndrome (RNS). At present, the pathogenesis of RNS are not entirely clear, clinical cure rate is low and lack of effective treatment, therefore, early diagnosis, effective treatment and judge prognosis for RNS become extremely important medicine field problems.Development of RNS is associated with multiple factors, studies confirm that RNS is associated with enhanced oxidative stress (ROS) reaction, ROS may participate in the glomerular podocytes, mesangial cells, endothelial cells, such as glomerular filtration barrier damage and subsequent process of the occurrence of proteinuria, and closely related to the severity of illness, so antioxidant treatment gradually attention by people. Thus, thioredoxin (TRX) and TXNIP play an important role in oxidative stress, is an important factor for the body to maintain redox balance. TRX are expressed in various body tissues and cells, it is by regulating cellular redox status against oxidative stress, thioredoxin-interacting protein (TXNIP) is one of the binding protein of TRX, by inhibiting the function of thioredoxin system and play a role of mediated oxidative stress. Therefore, TRX and TXNIP play an important role in oxidative stress, is an important factor of the body to maintain redox balance. RNS patients measure plasma TRX, TXNIP expression level, to explore the thioredoxin system in its role in the development of RNS patients. At the same time, micrornas (miRNA) has a variety of biological functions, highly conserved in evolution, plays an important role in the growth, physiological function, especially have significant effects on the occurrence of malignant tumor development. MiRNA compared with mRNA, more stable in vitro and in vivo metabolic cycle longer, they are involved in regulation of gene expression after transcription, via messenger RNA degradation or inhibition of protein synthesis.Many miRNA species control kidney cells in different pathological conditions, may be involved in the pathogenesis of kidney diseases. Study of miRNS in urine, large and stable, so in kidney disease, urinary micrornas have potential development prospects. Many RNS pathology exists in the extracellular matrix (ECM) protein accumulation, diffuse or nodular, glomerular sclerosis, tubular atrophy and interstitial fibrosis, miR-192is involved in the onset of the ECM process, and miR-192is the only one was found in glomerular mesangial cell expression of miRNA. At the same time, research hints in the transforming growth factor beta1(TGF-β1) induction of normal rat renal fibroblasts (NRK-49F) cell miR-29c expression by fibrosis process, while the expression of miR-29c enhancement can effectively inhibit TGF-β1and stimulate express of NRK-49cell lin Col Ⅰ, showed that miR-29c is inhibiting fibrosis genes. When or before in renal interstitial fibrosis, miR-29c high expression of cell can inhibit or delay the fibrosis progression. Therefore, correlation of miR-29c and RNS of renal interstitial fibrosis is particularly important. MiRNA always studies have shown that miR-29c, miR-192in kidney disease have different expression in peripheral blood and tissue, can be quantified in the urinary sediment, according to different pathological type urinary miRNA level is different, So the study of the miRNA can assist assessment of RNS pathological types, and judgement of the guidance of the clinical treatment and prognosis, and so on.At present, western medicine treatment of RNS is mainly hormone and immune inhibitors and cytotoxic drugs, but the side effects,limite clinical application. Therefore, developing the features and advantages of TCM, searching convenient and effective treatment of traditional Chinese medicine in RNS has important significance.RNS belongs to "edema","back pain","consumptive disease","urine turbid" and etc. in TCM."Edema" consistent with the RNS. Qi deficiency and blood stasis is the basis of RNS onset. Damp and hot accumulate knot is the catalyst in the process of development. The asthenia in origin and pathogenic factors in superficiality is the pathogenesis characteristics of RNS. So treatment with replenishing qi to invigorate the spleen and kidney and promoting blood circulation to remove blood stasis and clearing heat and promoting diuresisbeen detumescence, By astragalus, atractylodes, motherwort, leech, corn stigma, pyrrosia leaf of QZSZ particle comprise Huangqi,Baizhu,Yimucao,Shuizhi,Yumixu,Shiwei. According to the large sample study of RNS clinical, summed up the most often type of syndrome:qi deficiency and blood stasis and damp and hot, QZSZ particle become prescription for agreement of zhujiang hospital, southern medical university and TCM hospital in weihai city shandong province. QZSZ particle as an agreement prescription in hospital, has been used for many years, the clinical curative effect is verified, and can significantly reduce urinary protein, improve symptoms, reduce the high fat high coagulation state, delay the progress of glomerular sclerosis and protect renal function. But therapeutical mechanism of it in RNS is not clear, and needs further research. This topic through the treatment of QZSZ particle Detection clinical symptoms, blood biochemical indicators, serum superoxide dismutase (SOD), malondialdehyde (MDA), TRX and TXNIP ROS factor expression, provide the basis for clinical application. Through correlation study of gene expression of the urine miR-29c and miR-192and pathological types, it provide new targets for the diagnosis and treatment and prognosis of RNS.Chart Ⅰ QZSZ particle in the treatment of patients with RNS function and serum TRX, TXNIP expressionObjective:To observe the treatment of the QZSZ particle on qi deficiency blood stasis damp and hot syndrome in RNS patients, through testing the patient’s serum TRX and TXNIP expression, analysis of the two kinds of oxidative stress factor and RNS severity and prognosis of relevance, to explore its possible role in RNS development mechanism, which provide new targets for the treatment of RNS, and to study the relation of different clinical and pathological classification in curative effect.Methods:Choice of kidney disease center of zhujiang hospital, and TCM hospital in weihai in December2010to December2012outpatient or hospitalization RNS,60patients with clinical classification:22cases of often relapse,13cases of hormone dependence,2cases of hormone is invalid in,23cases of hormone some effects.46cases of whom underwent renal biopsy and pathological classification are:23cases of focal segmental glomerular sclerosis (FSGS),2cases of minimal change disease small lesion type (MCD),18cases of mesangial proliferative glomerulonephritis (MsPGN),3patients of membrane nephropathy (MN). randomly divided into30cases in experimental group (QZSZ particle plus foundation treatment) and30cases in control group (ambrette capsule plus and foundation treatment), treatment for3months, there is no difference between groups in age, sex, clinical classification, pathological classification, etc. Detection of two groups of curative effect of TCM syndrome score before and after the treatment, clinical efficacy,24h urinary protein, Alb, serum creatinine (Scr), prothrombin time (PT), partial prothrombin time (APTT), fibrinogen (FIB), the change of SOD and MDA. Using enzyme-linked immune detection technology (ELISA) to detect serum TRX and TXNIP expression level.Results:1. The relationships of pathological types and clinical classification:it was no significant difference (P>0.05); Relationships of clinical classification and pathological types of:FSGS and MsPGN ratio is higher, compared with other types have statistical significance (P<0.05).2. Two groups of clinical curative effect comparison:the total effective rate of experimental group compared with the control group, have statistical significance (P<0.05).3. Comparison of the curative effect of each clinical classification:there was no statistically significant difference (P>0.05).4. Comparison of the curative effect of each pathological types:curative effect of MN, MsPGN is obvious, it was statistically differences (P<0.05).5. The main syndromes of traditional Chinese medicine:the symptoms were improved after treatment, compared with before in two groups, treatment were statistically significant difference (P<0.01); The symptoms of comparative differences of two groups after treatment are statistically significant (P<0.01). the experimental group is better than the control group.6. Experimental group after treatment,24h urinary protein, Scr, BUN was significantly reduced, significantly reduced the difference was statistically significant (P<0.01); The control group after treatment compared with before treatment,24h urinary protein significantly reduced, significantly reduced,the difference was statistically significant (P<0.01); Compare the two groups after treatment, the experimental group in Alb to rise significantly, BUN dropped significantly, the experimental group were better than control group, the difference was statistically significant (P<0.05, P<0.01).7. Comparison of the experimental group before and after treatment,FIB decreased significantly, PT, APTT significantly increased (P<0.01), the control group before and after treatment comparison of PT,APTT significantly higher (P<0.01); Compare the two groups after treatment, the experimental group FIB decreased significantly, the difference was statistically significant (P<0.05).8. Comparison of the experimental group before and after treatment, a significant rise in SOD, MDA decreased significantly (P<0.01).Comparing the two groups after treatment, the experimental group than the control group SOD significantly increased, MDA decreased obviously (P<0.01).9. Comparison of the experimental group before and after treatment, TRX was significantly higher, TXNIP decreased significantly (P<0.05). Control group TRX, TXNIP comparison before and after the treatment, there was no statistically significant difference (P>0.05). Comparing the two groups after treatment, the experimental group than the control group TRX significantly increased, TXNIP decreased obviously (P<0.01).Conclusion:1. Proportion of FSGS and MsPGN in various clinical classification is higher. Efficacy of MCD, MsPGN in the pathological types of is higher.2.The QZSZ particle curative effect evaluation:can obviously drop of urine protein, is better than the control group.3. The relationship of the clinical classification and effect has proved that the curative effect of hormone dependence and often relapse is better, but there is no significant difference compared with hormone is invalid and some effects.The clinical has no obvious correlation with curative effect.4. The pathological classification relationship has proved that the effect of MsPGN and MCD is obvious, is better than with other pathological types.5. Comparitting curative effect of of QZSZ particle in TCM syndrome, the experimental group is better than the control group, after treatment the total integral of TCM symptoms decreased obviously, the experimental group is better than the control group. QZSZ particle can heal RNS better in TCM symptoms.6. QZSZ particle significantly elevated plasma Albumin, protect the renal function.7. QZSZ particle can improve high blood coagulation state in patients with RNS, rise in PT and APTT, reduce FIB was significantly better than the control group.8. QZSZ particle can increases SOD level and reduce the MDA level of the RNS patients, improve the body state of oxidative stress.9. QZSZ particle can increase TRX levels, lower levels of TXNIP. TRX, TXNIP as oxidative stress related factors, play a role in the RNS.10. QZSZ particle may play an important role in the treatment of RNS through the mechanism of resistance to oxidative stress.Chart II Preliminary study of expression of the urinary miRNA-192,29c with curative effect of QZSZ particle inRNS and pathology of RNSObjective:Study internal relation of urine miR-192and miR-29c in different clinical pathological types, explore the urine miR-192and miR-29c to distinguish the severity of disease and renal histological lesions of the possibility as a non-invasive biomarkers. Urinary miR-192and miR-29c expression of before and after QZSZ particle treatment are associated with24h urinary protein, Alb, TC, Lp-a and so on.Methods:Selection of kidney disease center of zhujiang hospital, southern medical university and nephrology department in TCM hospital in weihai in December2010to December2012outpatient or hospitalization of the renal biopsy diagnosed44RNS patients, including18cases of MsPGN,3cases of MN and23cases of FSGS, randomly divided into the experimental group(QZSZ particle plus foundation treatment) and the control group,21cases of control group (ambrette capsule plus foundation treatment), treatment for3months, each group in age, gender, pathological classification, no difference. Set up15healthy volunteers as healthy controls in medical staff weihai hospital. Before and after3months of treatment RNS patient t collect specimens of blood and urine samples of100ml early in the morning on an empty stomach, healthy people to collect urine samples of100ml early in the morning on an empty stomach, urine specimen in3000RMP centrifugal before30minutes, again in13000RMP centrifuge for5minutes, discarding the supernatant fluid, the urine sediment repackaging in1.5ml EP tube,0.25ml per tube. Add0.75ml per EP tube Trizol LS, volatility1min cracking fully, let stand at room temperature after5min-80℃refrigerator. Trizol to extract total RNA, RNA quality testing after qualified, can build miR-192and miR-29c RT-PCR reaction system, each sample was detected in miR-192and miR-29c. The relative expression quantity and do statistical analysis. Test24hours urinary protein, Lp-a,TC, Alb before and after the treatment, test correlation of the urine miR-192and miR-29c and the above indexes.Results:1. Compared with healthy controls, miR-29c expression in the FSGS decreased obviously (P<0.05), the expression of MsPGN, MN decline is not obvious (P>0.05). miR-192in MsPGN expression increased obviously (P<0.05), the expression of MN,FSGS is not obvious (P>0.05).2. The expression of urinary miR-29c and Lp-a showed a negative correlation, It was positively correlated with Alb, had statistical significance (P<0.05). The expression of urinary miR-192express positive correlation with the expression of urinary24h urinary protein and TC, had statistical significance (P<0.05).3. Comparison of the experimental group before and after the treatment, the expression of miR-192significantly reduced and the expression of miR-29c significantly rised (P<0.01). Comparison of the control group before and after the treatment, the expression of miR-192significantly reduced and the expression of miR-29c significantly rised (P<0.05).Comparison of two groups after treatment, the expression of miR-29c in the experimental group significantly rised (P<0.05)Conclusion:1. Urine miR-192expression significantly increased in MsPGN patients.2. MsPGN patients may have more ECM secretion and deposition, can determine glomerular ECM. It can guide clinical treatment and prognosis.3. The urine miR-29c expression in patients with FSGS decreased significantly.4. Patients with FSGS may be renal interstitial fibrosis significantly. It can judge the degree of renal fibrosis, increase urine levels of miR-29, help in treatment of renal fibrosis.5. Differences in expression of urine miR-192,29c shows the differences between the various pathological types in RNS, and provides new ways to better understand the pathogenesis of RNS.6. The expression of urine miR-192,29c with24h urinary protein, TC, Lp-a shows that several factors can increase renal fibrosis and ECM deposition.7. QZSZ particle can increase the expression of urine miR-29c levels, and lower the expression of urinary miR-192levels in patients, play a regulatory role of the removal of the ECM, anti fibrosis, may through the two-way function to inhibit proliferation of ECM, delay the glomerular sclerosis.