| ObjectiveRenal interstitial fibrosis is chronic kidney disease (Chronic kidney disease, CKD) is an important development and prognostic factors, has the vital significance of early prevention and reversal of renal interstitial fibrosis to prevent the development of CKD. The gold standard for diagnosis of renal disease renal biopsy, but it is an invasive operation, need to take out a small part of renal tissue pathology, there are3%main complications such as bleeding, tissue injury risk, so looking for a reliable, noninvasive biomarkers has important significance.MicroRNAs (miRNAs) plays an important role in the progression of renal disease diagnosis and diagnosis of disease, miRNAs are very stable in tissues and fluids, urine collection operation is simple and feasible, so the identification of urinary miRNAs level can be used to judge the disease a potentially attractive biomarker. MiR-21is a key link in renal fibrosis, sustained overexpression of miR-21disrupted the tissue repair and cause tissue fibrosis, the research reported in renal fibrosis in unilateral ureteral obstruction (UUO) animal model, the expression level of miR-21increased, the degree of renal fibrosis and inhibition of miR-21expression after rat model was improved. Inhibition of miR-21is a new way to improve the tissue fibrosis.In recent years the research on Modern Chinese medicine is urgent needs to have the characteristics of TCM syndrome or "combination of disease and syndrome" biomarkers and animal model, in order to make the research work of traditional Chinese medicine is more scientific, objective, this is currently the hotspot and difficulty in study of Chinese and Western medicine ". Reports in the literature, Qi deficiency and blood stasis syndrome of renal interstitial fibrosis in patients with the most common syndromes of traditional Chinese medicine, the clinical study, quantitative polymerase chain reaction (RT-qPCR) expression in renal interstitial fibrosis of renal biopsy in the patients with Qi deficiency and blood stasis in urinary sediment in the determination of miR-21mRNA, and to explore its correlation with renal degree, interstitial fibrosis of qi deficiency and blood stasis score of.Previous studies have suggested three Sanqi oral liquid (preparation, Guangdong Province Traditional Chinese Medical Hospital hospital from Astragalus, three seven etc.) can effectively prevent the unilateral ureteral obstruction (UUO) rat model of renal interstitial fibrosis, but the specific mechanism needs further study. In this study, we through the model of unilateral ureteral obstruction (UUO) rat fatigue exhaustive swimming method to establish the model of combination of disease and syndrome (renal interstitial fibrosis filling deficiency and blood stasis model), miR-21mRNA in renal tissue was detected by gene chip detection and three Sanqi oral liquid with differential gene expression, further validated by RT-qPCR, to illustrate three Sanqi oral liquid for prevention of UUO model rats with Qi deficiency and blood stasis of renal interstitial fibrosis mechanism.In summary, this study aimed at the expression of renal interstitial fibrosis of miR-21mRNA was discussed, the correlation between clinical part aims at detecting renal interstitial fibrosis in patients with Qi deficiency and blood stasis of urine miR-21mRNA expression and discussion and the degree of fibrosis, in order to explore non-invasive biomarkers for this disease to provide basis for Forecasting Experiment aims to build; the foundation of interstitial fibrosis filling deficiency and blood stasis syndrome of kidney, detection of expression of miR-21mRNA in renal tissue and gene chip detection three Sanqi oral liquid stem gene expression difference in prognosis, in order to explain the three Sanqi oral liquid for prevention of UUO model rats with Qi deficiency and blood stasis of renal interstitial fibrosis mechanism. Methods1clinical study1.1case selection Selected in accordance with the inclusion criteria (syndrome differentiation standard is in accordance with the diagnostic standard of chronic kidney disease and syndrome of qi deficiency and blood stasis, except) and excluded patients with a total of30cases.1.2research groupsBe admitted to hospital after renal biopsy, according to renal pathology suggesting there is no renal interstitial fibrosis and divided into fibrosis group and no fibrosis group two groups. Fibrosis group renal pathology suggests the presence of renal interstitial fibrosis in patients with a total of20cases, no renal interstitial fibrosis in patients with a total of10cases of healthy control group; the other10cases.1.3detection indicators and research contentDetection of two groups of patients before kidney biopsy24h quantitative urinary protein, hemoglobin, serum albumin, serum creatinine, calculated glomerular filtration rate (eGFR) integration and calculation of qi deficiency and blood stasis; get on and healthy group of renal biopsy in patients with morning urine, using quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-21mRNA urinary sediment differences between groups were compared, the expression of urine miR-21mRNA, and to explore its and renal interstitial fibrosis, Qi deficiency and Blood Stasis Syndrome Correlation between indicators such as.2experimental study2.1research groups24SD rats were randomly divided into three groups:control group, fibrosis of qi deficiency and blood stasis model group (hereinafter referred to as model group), fibrosis of qi deficiency and blood stasis+Sanqi oral liquid group (hereinafter referred to as the Sanqi group), each group of8only.2.2modeling methods2.2.1model of UUOThe UUO model with left ureteral ligation, preoperative disinfection of equipment, rats with10%chloral hydrate intraperitoneal injection anesthesia of0.30.4ml/100g, will be fixed in the prone position in the rat board, shaving, regular disinfection, wearing sterile gloves, Pu aseptic hole towel, in rat dorsal part, the left rib waist point about0.5cm, the spine on the left about lcm as a longitudinal incision,1.5～2cm long, layer by layer separation of subcutaneous tissue, muscle, full exposure of the kidney, with a small curved forceps gently peel kidney and lower portion of the capsule and renal pedicle of envelope, isolated ureter, with "1" Silk difference in close proximity to the pelvis at and far from the renal Muang yo lcm ligation of the ureter, two in middle ureter node disconnection, peritoneal lavage with gentamicin saline, check no leakage and hemorrhage after sutured back muscle and routine disinfection.2.2.2model of qi deficiency and blood stasisTCM syndrome model rats by exhaustive swimming fatigue replication of qi deficiency and blood stasis syndrome, model of replication for28days. Swimming conditions:(1):24～25℃at room temperature;(2):22～24℃swimming water temperature;(3) the swimming venue:diameter50cm, height60cm circular water tank, water depth control for45-50cm;(4) exhaustive standard: the rat sank10s cannot be self surfaced as exhaustive, coming out in rats. Stop when all of the50%natural subsidence.2.2.3fibrosis of qi deficiency and blood stasis model replicationAccording to the literature reports and previous experimental results, replication method using UUO model rat fatigue exhaustive swimming experiment: in the UUO model for10days, rats wound healed completely after the start of fatigue after exhaustive swimming, for28consecutive days model replication.SanQi group intervention start time and Qi deficiency and blood stasis model copy, time synchronization, the morning model copy, the afternoon to drug intervention. Three Sanqi oral liquid group according to the adult daily dose calculation in rats daily dose, daily rats at doses of1.2ml/100g rats, while other groups given equal volume of distilled water, continuous administration for28days.2.3intervention measuresSanQi group intervention start time and Qi deficiency and blood stasis model copy, time synchronization, the morning model copy, the afternoon to drug intervention. Three Sanqi oral liquid group according to the adult daily dose calculation in rats daily dose, daily rats at doses of1.2ml/100g rats, while other groups given equal volume of distilled water, continuous administration for28days.2.4indexes2.4.1rats were detected by HE in renal tissue, Massonstaining and AFS, the pathology of CIS integral, integralchange of qi deficiency and blood stasis syndrome.2.4.2gene chip detection:specific steps, total RNA in renal tissue of the further purification, identification and labeling; RNA identification; rats using Agilent microarray gene expression profiling (each chip contained41000gene probe), each labeled RNA samples were individually with a gene chip hybridization (each time point experimentgroup, control group5specimens). Statistical analysis:using data processing software of Agilent microarray dataextraction software10.5.1.1for processing the results ofscanning; analysis of differential gene analysis and signalpathway is the experimental group of two independent samples t test and control group, P<0.05, gene expression or tone ratio≥2as the standard, there was significant difference is the signal transduction genesassociated with fibrosis. Signal pathway analysis with Fisher’s exact test, P<0.05had significant difference.2.4.3quantitative polymerase chain reaction (RT-qPCR) detection of gene groups and the difference of the expression of miR-21mRNA three, to explore the mechanism of Sanqi oral liquid on renal interstitial fibrosis model of qi deficiency and blood stasis rats.Results1clinical study1.1participants in general30patients met the inclusion and exclusion criteria of patients with chronic kidney disease, including16cases of male, female14cases. Another selection of healthy group10cases, including6males,4females. Chronic kidney disease group and healthy group in gender, age, there was no significant difference between;1.2patientsRenal biopsy that after admission,24patients with IgA nephropathy,3cases of focal hyperplasia of glomerular sclerosis,3cases of chronic interstitial nephritis; there were20cases of interstitial fibrosis in patients with renal interstitial fibrosis,10cases of patients without kidney. The renal biopsy suggested the presence of renal interstitial fibrosis in20cases were included in fibrosis group, no renal interstitial fibrosis in patients with10cases into no fibrosis group;1.3The degree of fibrosis and biochemical indices of renal mass points, Qi deficiency and blood stasis syndrome.Fibrosis group and no fibrosis group, Qi deficiency and blood stasis syndrome score was significantly increased, with significant difference (P<0.05), two groups were comparable in terms of age, hemoglobin, serum albumin,24h urinary protein, serum creatinine, eGFR and so on, there were no significant differences in;1.4The expression level of miR-21mRNA in urinary sedimentFibrosis group compared with no fibrosis group patients and healthy controls, the expression level of miR-21mRNA in urine were significantly increased, with significant difference (P<0.05, P<0.01); no fibrosis patients compared with healthy controls, there was no significant difference in;1.5correlation studyRenal interstitial fibrosis and urine levels of miR-21mRNA, Qi deficiency and blood stasis syndrome score was positively correlated (correlation coefficients were r=0.573, p=0.001; r=0.531, p=0.003); negative correlation with eGFR (correlation coefficient r=-0.620, p=0.000); Urine miR-21mRNA levels and the degree of interstitial fibrosis, kidney qi deficiency and blood stasis syndrome score was positively correlated (correlation coefficients were r=0.573, p=0.001; r=0.619, p=0.000).2experimental study2.1in generalIn the course of the experiment, the model group and three rats of group1rats died due to drowning, swimming fatigue experiment, the blank control group weight growth significantly, in good spirits, ease, sensitive reaction, fur shiny; rats in the model group was thin, drink more water, urine, stool pond, apathetic, reflect the slow, gross vertical dull, slow. San Qi group rats were in poor mental health status, the action is slightly slow, form a model group increased, more urine, stool forming, to reflect the sensitivity was worse than that of normal group is better than that of the model group.2.2Qi deficiency, blood stasis score2.2.1compared with the control group, the rats in the model group, Qi deficiency blood stasis score increased significantly, the difference was statistically significant (P<0.05, P<0.01), suggesting that the model group the successful construction of "Qi deficiency and blood stasis syndrome" model.2.2.2compared with the model group, three rats in group of qi deficiency syndrome, blood stasis score were significantly reduced, the difference was statistically significant (P<0.05), and compared with the control group, no significant difference. The results suggest that the three Sanqi oral liquid can improve the effect of Supplementing Qi and activating blood circulation in the rat model of qi deficiency and blood stasis syndrome.2.3HE and Masson staining, CIS and AFS scoreHE, Masson staining, obvious renal interstitial fibrosis model group, Astragalus group three, CIS, AFS score increased significantly, compared with the control group, with significant difference (P<0.001), renal interstitial fibrosis model was constructed successfully; San Qi group compared with model group, model rats kidney interstitial fibrosis significantly improved, CIS, AFS score was significantly decreased, there was significant difference (P<0.05).2.4the results of gene chipSignal pathway analysis:model group compared with the control group, a total of42signal pathway up-regulated, with significant difference (P<0.05), the MAPK signaling pathway and fibrosis related Fisher-Pvalue was0.0058; San Qi group compared with model group, a total of30signal pathway is down regulated, with significant difference (P<0.05), where MAPK signal pathway of P value is0.013.Differential gene expression analysis:model group compared with the control group, increased MAPK signaling pathway of p38/MAPK signal pathway in multiple genes, with significant difference, especially the proinflammatory cytokines, such as TNF, IL-1, FASL, up-regulated the expression of TGF-β,with significant difference (P<0.05); San Qi group compared with model group, down-regulation of multiple genes ERK/MAPK and p38/MAPK in the MAPK pathway, the differences were significant, especially the proinflammatory cytokines, such as TNF, IL-1, down regulated the expression of FASL, with significant difference (P<0.05)2.5Expression of TNF-α, IL-1RT-qPCR, miR-21, Smad7, α-SMA mRNAAccording to the gene chip results and literature reports, the use of RT-qPCR for detection of TNF-a expression, IL-1, miR-21, Smad7,α-SMA mRNA. The model group compared with the control group, the expression of IL-1, miR-21, α-SMA mRNA were significantly increased (P<0.01, P<0.05); Sanqi group compared with model group, the above three indexes were significantly lowered (P<0.05). Compared with the blank group, model group and Astragalus group three expression of Smad7mRNA is elevated, San Qi group compared with the control group, the difference was statistically significant (P<0.05), but the difference was not statistically significant in model group and blank group. No statistically significant differences in the expression of TNF-α mRNA in three groups.Conclusion1clinical study1.1Expression levels of renal interstitial fibrosis in patients with Qi deficiency and blood stasis of urine miR-21mRNA was significantly higher than in healthy group and no fibrosis group, the difference was statistically significant (P<0.05, P<0.01); no renal interstitial fibrosis patients compared with healthy controls, no significant difference.1.2urinary sediment miR-21mRNA levels and the degree of interstitial fibrosis, kidney qi deficiency and blood stasis syndrome score was positively correlated (correlation coefficients were r=0.573, p=0.001; r=0.619, p=0.000). These results suggest, urine miR-21mRNA may be one of the specific indicator of renal interstitial fibrosis in patients with Qi deficiency and blood stasis, worthy of further enlarging the sample of in-depth research in the future.2experimental study2.1model of unilateral ureteral obstruction (UUO) and fatigue exhaustive swimming test model, the pathological results showed that renal tubulointerstitial fibrosis model group obviously, Qi deficiency and blood stasis score significantly increased, renal tissue miR-21mRNA expression was up-regulated, suggesting that successfully reproduced "renal interstitial fibrosis and Qi deficiency and blood stasis syndrome""combined with" animal model.2.2three Sanqi oral liquid can improve UUO rats with Qi deficiency and blood stasis of renal interstitial fibrosis, and its mechanism might be through downregulation of miR-21, IL-1,α-SMAmRNA, upregulation of Smad7mRNA levels, improve the inflammatory state, reduce fibrosis cell proliferation, thereby improving the renal interstitial fibrosisIn summary, this study from the clinical and experimental research for the two part of the expression of miR-21in renal interstitial fibrosis was explored, provides a reliable, non-invasive clinical predictors for renal interstitial fibrosis in patients with Qi deficiency and blood stasis, and explains the mechanism of Sanqi oral liquid three effective prevention and treatment of renal interstitial fibrosis of qi deficiency and blood stasis, laid the foundation for the further development of new drugs. |
PDF Full Text Request