BMSCs-derived exosomal microRNA-let-7a plays a protective role in diabetic nephropathy via inhibition of USP22 expression.

OBJECTIVE: Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease. The molecular pathogenesis of DN is still poorly understood. This study was designed to investigate the protective effect of bone marrow mesenchymal stem cell (BMSCs)-derived exosome (Exo)-transported microRNA-let-7a (miR-let-7a) on DN by targeting ubiquitin-specific protease 22 (USP22). METHODS: BMSCs of rats were cultured, and exosomes were identified. A rat models of DN were established in this study, and the rats were injected with Exo, Exo-let-7a mimic, or si-USP22 to figure their functions in renal function indicators blood urea nitrogen (BUN) and serum creatinine (SCr), blood lipid-related indicators total cholesterol (TC) and triglyceride (TG), renal cell apoptosis, oxidative stress, and expression of N-cadherin and vimentin in renal tissues. MiR-let-7a and USP22 targeting relationship was validated. RESULTS: Suppressed miR-let-7a and over-expressed USP22 exhibited in renal tissues of DN rats. Exosomes increased miR-let-7a and repressed USP22 in renal tissues of DN rats. Moreover, elevated exosomal miR-let-7a or silenced USP22 reduced SCr, BUN, TG and TC, suppressed apoptosis of renal cells and oxidative stress, and inhibited N-cadherin and vimentin expression in renal tissues of DN rats. MiR-let-7a had a targeting relationship with USP22. CONCLUSION: Our study highlights that BMSCs-derived exosomal miR-let-7a represses renal cell apoptosis, which plays a protective role in DN through down-regulation of USP22.

[Identification of differentially expressed genes in rats and preliminary analysis in regression of vascular calcification].

OBJECTIVE: To investigate the differentially expressed genes in rat in the process of regression of vascular calcification by using the suppression subtractive hybridization (SSH). METHODS: 24 SD male rats which aged 6 weeks and specific pathogen free grade were selected and randomly divided into 3 groups (n = 8): control group, calcification group and regression group respectively. Vascular calcification model (vitamin D3 plus nicotine, VDN) were made from rats in calcification group and regression group, and rats in control group were intragastric administered with normal saline and lavaged with peanut oil. Rats were bred for 8 weeks in calcification group and control group, while rats in regression group were fed for 16 weeks. All rats were killed to measure concentration of calcium in the arterial tissue and examine the pathological lesion changes. Subtractive hybridization among vascular cDNA sequences from calcification group and regression group were established. The cDNA fragments which expressed higher or lower in regression group than those in calcification group were isolated. Differentially expressed genes with cDNA fragment were inserted into PMD18-T plasmid vector and transformed competent DH-5alpha, cDNA libraries of differentially expressed gene between calcification group and regression group were then constructed. Recombinant vectors were analyzed by colony PCR, positive genes were randomly selected for sequencing and analyzed by BLAST. 4 genes were randomly selected for RT-PCR certification combined with semi-quantitative analysis of DNA bands. RESULTS: VDN model of rats were successfully constructed. Concentration of tissue calcium in calcification group (15.34 mg/g +/- 2.51 mg/g) was significantly increased compared to that in control group (5.20 mg/g +/- 0.75 mg/g, P < 0.001), while in comparison with calcification group (15.34 mg/g +/- 2.51 mg/g), calcium in regression group was relatively lower (12.73 mg/g +/- 1.89 mg/g, P < 0.05). 28 up-regulated genes and 22 down-regulated genes were gained through sequencing and BLAST analysis among positive clones. RT-PCR validation indicated that 4 genes such as prdx3 and Ank2 had increasedly expressed in regression group than those in calcification group, the average fold change was 1.7. CONCLUSION: Rat vascular calcification tissue had characteristic of active regression. Genes in relation to pyrophosphoric acid synthesis, glutamate signal peptides, anti-oxidant and ant-apoptosis were up-regulated, at the same time many genes related to ossification and oxidation activity were down-regulated in the process of calcification regression. Increased expression of calcification suppressor genes accompanying decreased expression of calcification promoting genes might be the intrinsic mechanisms which initiated the active regression of calcified tissues.

[Gastrodine represses expression of IL-1 beta, IL-6 induced by hyperglycemia in gitter cells].

OBJECTIVE: Inflammatory factors have been known to induce nerve cells apoptosis and decrease learning capacity of diabetics. The aim of this study is to evaluate the inhibitory effect of Gastrodine on the expression of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) in culturing for gitter cells (BV-2 cells) induced by high concentration of glucose. METHOD: The BV-2 cells incubated in vitro with different concentrations of glucose and gastrodine were divided into five groups: control group (glucose: 25 mmol x L(-1)), high concetration of glucose (glucose: 45 mmol x L(-1) HCG) group and Gastrodine groups (glucose 45 mmol x L(-1) with gastrodine 25 mg x L(-1) (LG), 50 mg x L(-1) (MG), 100 mg x L(-1) (HG). After culturing for 24 h, morphological changes of cells were observed by inverted phase contrast microscope. The supernatant protein of IL-1 beta and IL-6 was detected by ELISA. The mRNA expression of IL-1 beta and IL-6 was assessed by Reverse transcription polymerase chain reaction (RT-PCR). RESULT: The cells were proned to aggregate, some of them with hypertrophy, distinct nucleoli and branch-shaped hyperplasy in HCG group, while less change in Gastrodine groups. The supernatant protein of IL-1 beta is higher in HCG group than control group (119.53 +/- 15.91) ng x L(-1) vs (25.74 +/- 15.72) ng x L(-1) (P < 0.01), but lower in the gastrodine groups than HCG LG (99.32 +/- 19.66) ng x L(-1), MG (76.94 +/- 17.16) ng x L(-1), HG (88.35 +/- 18.72) ng x L(-1) vs (119.53 +/-15.91) ng x L(-1) (P < 0.05). The supernatant protein of IL-6 protein also higher in HCG than control group (393.7 +/- 17.51) ng x L(-1) vs (125.85 +/- 36.62) ng x L(-1) (P < 0.01), and lower in the gastrodine groups than HCG (LG 327.06 +/- 23.53) ng x L(-1), MG (217.36 +/- 28.81) ng x L(-1), HG (263.17 +/- 22.32) ng x L(-1) vs (393.7 +/- 17.51) ng x L(-1), P < 0.05). The mRNA expression of IL-1 beta was increased significantly higher in HCG than control group (2.77 +/- 0.29) vs (1.13 +/- 0.27) (P < 0.05), but decreased significantly in gastrodine groups than HCG LGA (2.66 +/- 0.31), MGA (2.1 +/- 0.41), HGA (2.4 +/- 0.28) vs (2.77 +/- 0.29) (P < 0.05). The mRNA Expression of IL-6 was higher in HCG than control group (3.97 +/- 0.33) vs (1.05 +/- 0.13) (P < 0.05, but lower in gastrodine groups than HCG LG (3.28 +/- 0.3), MG (2.65 +/- 0.33), HG (3.04 +/- 0.26), vs (3.97 +/- 0.33) (P < 0.05). CONCLUSION: Gastrodine can inhibit the expression of IL-1 beta, IL-6 in cultured BV-2 cells induced by high concentration of glucose.