| Objective1. Clinical StudyTo study the distribution of age and gender, clinical type, pathological type and activity index in lupus nephritis patients. To study differences between sthenia type and deficiency type of TCM by comparing above indicators and other clinical indicators, immune indicators, inflammation indicators. To provide objective basis for treatment based on syndrome differentiation through analyzing the correlation with activity of this disease between the two types.2. Experimental StudyTo study differences of microRNA expression profiles in plasma exosome between spleen kidney yang deficiency type of lupus nephritis and healthy volunteers. To find out target gene which was predicted by the differentially expressed molecules and then search for their biological pathways and the main functions of them to study the possible mechanism of lupus nephritis; Trying to find out some differentially expressed microRNA molecules as biomarkers that can be good used in the diagnosis of the disease; To study the correlation between the specific expression of microRNA molecules and spleen and kidney yang deficiency syndrome in lupus nephritis, and in order to provide an objective basis for syndrome differentiation of TCM.Methods1. Clinical StudyA retrospective survey was done with inpatients of lupus nephritis from January 2010 to December 2015. These patients all came from renal internal department in the First Affiliated Hospital of Guangzhou University of Chinese Medicine. They were selected according with diagnostic criteria, inclusion criteria and exclusion criteria. Their types were judged by signs and symptoms when they were checked by renal biopsy for the first time. They were divided into five types, including heat toxin syndrome, yin deficiency of liver and kidney syndrome, two deficiency of Qi and yin syndrome, qi deficiency of spleen and kidney syndrome, yang deficiency of spleen and kidney syndrome. We divided the first type into sthenia type, and the other four types into deficiency types. We collected information of age and gender, clinical types, pathological types, activity index, immune indicators and clinical physicochemical indicators. We analyzed and compared differences between the two groups through above indicators by using SPSS software.2. Experimental StudyTo select yang deficiency of spleen and kidney type in lupus nephritis patients and healthy volunteers, each group had three cases. We extracted exosome of plasma with Exoquick reagent, and then checked the microRNA expression profiles of exosome in two groups by high-throughput sequencing method. We screened differential expressions of microRNA molecules and predicted their target genes. To summary the main gene functions and signaling pathways by analysing the KEGG biological pathway and GO function of the target genes.Results1. Clinical StudyThis study collected a total of 134 patients. There were 117 female patients and 17 male patients. The ratio of male to female was about 1:6.9. The most common type of TCM was deficiency of spleen and kidney syndrome. It had a total of 41 cases and accounted for about 30.6%; The second type was heat toxin syndrome and Yang deficiency of Spleen and kidney syndrom. Each type of them had 33 patients and accounted for about 24.63%; The following was yin deficiency of liver and kidney syndrome and had 14 cases (10.45%). The next was deficiency of Qi and yin syndrom and had 13 cases (9.70%).The group of sthenia type had 33 cases and deficiency type had patients with 101 cases.(1) General data of gender and age:There were only 2 cases of male patients and 31 cases of female patients in sthenia group;Their ages ranged from 12 to 45 years old. The average age was (27.76+10.80) years old. There were 15 cases of male patients and 86 cases of female patients in deficiency group;The minimum age was 10 years old and the maximum age was 60 years old. The average age was (30.68+12.07) years old. The differences of age and gender between two groups were not statistically significant. LN patients had no significant correlation with gender and age in two groups.(2) Clinical types:The most common type in clinical classification was nephrotic syndrome. This type had 59 cases (44.03%);The second type was chronic glomerulonephritis. This type had 48 cases (35.82%);The next type was latent glomerulonephritis and it had 12 cases (8.96%);The next was chronic renal failure type. This type had 11 cases(8.21%);The least was type of rapidly progressive glomerulonephritis.It only had 4 cases (2.98%). The comparison of clinical classification between two groups did not have statistically significant differences. The most commom type in sthenia syndrome was chronic nephritis, but it was nephropatic syndrome in deficiency syndrome.Rapidly progressive glomerulonephritis was only found in deficiency syndrome. Its main type of TCM was Yang deficiency of spleen and kidney syndrome.(3) Pathological indicators:The most common pathological type was IV. This type had a total of 50 cases (37.31%);The second type was type V and had 23 cases (17.16%);The next was V+IV type and had 20 cases (14.93%);The following was type II and had 17 cases (12.69%), type I had 12cases(8.96%) and type III had 7 cases (5.22%), type V+III had 3 cases (2.24%), type VI had 2 cases (1.49%). The comparison of pathological type distribution and AI between two groups did not have statistically significant differences. The CI in sthenia type was higher than that in deficiency type.There was statistically significant differences between them.(4) Clinical physicochemical indicators:There were statistically signif-icant differences of some indicators which included WBC, LYM, RBC and HGB in routine test of blood between two groups. The counts of WBC and LYM in deficiency type were significantly higher than sthenia type,but counts of RBC and HGB were significantly lower than those in sthenia type. There were also statistically significant differences in indicators of renal function which included BUN, UA and cystatin C. The comparison of creatinine had no statistical difference between two groups. The levels of BUN, UA and CysC in deficiency type were significantly higher than those in sthenia type;There were also statistically significant differences in indicators of liver function which included AST between two groups.The differences of ALT, ALB and G were not statistically significant. The level of AST in deficiency type was significan-tly higher were than than in sthenia type.(5) Immune indicators:ANA were positive in all patients. The total of ds-DNA positive patients were 113 cases (84.33%). In anti-ENA spectrum, the most common was with anti-SSA antibody positive. It had a total of 79 cases (58.96%);The second was anti-Sm antibody positive and had 77 cases (57.46%). The next was anti-nucleosome antibody (Anua) positive and had a total of 73 cases (54.48%).The following was anti-histone antibody (AHA) positive and had a total of 58 cases (43.28%). Anti-nRNP antibodies positive patients were 42 cases (31.34%).Anti-rRNP antibody positive patients were 38 cases (28.36%). Anti-SSB antibody positive patients were 34 cases (25.37%). Anti ScL-70 antibody positive were only 5 cases (3.73%). Anti JO-1 antibody positive patients only had 1 cases (0.75%) and anti centromere antibody positive patients did not be found. The comparison of the antibody positive rate between two groups did not have statistically significant;The comparison of the five immune indicators including ANA titers, dsDNA titer, CIC, C3 and C4 also did not have statistically significant differences between two groups.(6) Inflammation indicators:Because some patients did not test CRP and ESR, so the actual statistics of the sthenia group were 20 cases and a total of 64 cases in deficiency group;The comparison of CRP and ESR between two groups did not have statistically significant differences.(7) SLEDAI score:In all of the 134 patients, the highest SLEDAI score was 40 points and the lowest was 2 points. The average score was (13.39+0.48) points. There were 62 cases(46.27%) which was in severe active stage of this disease. There were 36 cases(26.86%) which was in moderate active stage and 28 cases (20.9%) in mild active stage. There were only 8 cases (5.97%) of inactive stage.The severe active stage was the most common in both of two groups;The second was moderate active stage;The next was mild active stage;The inactive stage was the least. There were no statistically significant differences about SLEDAI scores and its stage between two groups.(8) Urine indicators:In all of the 134 patients,128 cases of them were with positive urine protein. It accounted for 95.52%;The most common condition was 3 plus of proteinuria and had a total of 95 cases(70.9%);There was only 6 cases whose urinary protein were negative. A total of 115 cases were with urine blood positive and accounted for 85.82%; The most common condition was also 3 plus of urine blood and had a total of 77 patients(57.46%);There was only 19 cases whose urine blood were negative. The comparition about qualitative of urine protein, urine blood and quantitation of urine protein between two groups did not have statistically significant differences.2. Experimental Study(1) Compared the miRNA expression profiles in serum exosomes between yang deficiency of spleen and kidney syndrome in lupus nephritis and healthy volunteers;As a result,148 differentially expressed miRNA molecules were selected. The number of the up-regulated expression was 131 and down-regulated expression was only 17. There was a total of 2 cases which had high significant differences. They were hsa-miR-4497 and hsa-miR-127-3p; There was a total of 6 cases which had moderate significant differences. They respectively consisted of hsa-miR-129-5p, hsa-miR-383-5p, hsa-miR-423-5p, hsa-miR-629-3p, hsa-miR-3184-3p and hsa-miR-3158-3p;The remaining of 140 cases had only general differences.(2) We screened a total of 1664 target genes through eight miRNA molecules whose expressions were significant differences. The number of the target genes for hsa-miR-629-3p was the most and had a total of 439. The least was the target genes for hsa-miR-127-3p and only had 8 genes. Hsa-miR-3158-3p was a miRNA molecule of down-regulated expression and the target genes for it were a total of 205.(3) By analyzing target gene through KEGG pathway, we get a total of 19 signaling pathways and disease pathways which were statistically significant. The more significant pathway was FoxO signaling pathway and TGF-signaling pathway in those of them.(4) We get a total of 108 gene functions by analyzing target genes in GO database. Their functions were mainly embodied in the aspects of cell autophagy and regulation gene expression, biosynthesis, transcription factor activity and so on.Conclusion1. Clinical Study(1) The most common type of TCM is deficiency of spleen and kidney in lupus nephritis patients which includes both qi deficiency of spleen and kidney syndrome and yang deficiency of spleen and kidney syndrome;The most common type in clinical classification is nephrotic syndrome. The second type is chronic nephritis;The most common type of pathology is type Ⅳ.The following types are type V and type V+IV; The most common stage in this disease is severe active stage according to SLEDAI score.(2) LN patients have no significant correlation with gender and age between sthenia group and deficiency group.(3) There are no significant differences in clinical classification, pathological type, five immune indicators, anti-ENA spectrum, SLEDAI score, inflammation indicators and urine indicators between sthenia group and deficiency group. There is no significant correction with activity of the disease between two groups.(4) The indictors including WBC, LYM, BUN, UA, CysC and AST in deficiency group are significantly higher than those in sthenia group, but the levels of RBC and HGB are significantly lower than those in sthenia group. The results show that the degrees of the renal function damage and anemia in deficiency group are heavier than those in sthenia group;The degree of renal chronic damage in deficiency group is heavier than that in sthenia group.2. Experimental Study(1) Hsa-miR-127-3p and hsa-miR-4497 molecules in yang deficiency of spleen and kidney syndrome are significantly expressed in lupus nephritis patients and may play an important part in the diagnosis and syndrome differentiati-on, but it need do further study to verify the opinion.(2) FOXO signaling pathway and TGF beta signaling pathway may play an important role in the pathogenesis of LN. The abnormalities of cell autophagy and the regulation of gene expression, biosynthesis, transcription factor activity function may be the important pathogenesis of LN, but it need to be confirmed by further researches. |
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