Critical Limb Ischemia (CLI) is common but uncommonly diagnosed. Improved recognition and early diagnostic markers for CLI are needed. Therefore, the aim of our study was to identify plasma biomarkers of CLI in patients with type 2 diabetes mellitus (T2DM). In this study, antibody-coated glass slide arrays were used to determine the plasma levels of 274 human cytokines in four matched cases of diabetes with and without CLI. Potential biomarkers were confirmed in an independent cohort by ELISA. After adjusting for confounding risk factors, only plasma level of Siglec-5 remained significantly associated with an increased odds ratio (OR) for diabetes with CLI by binary logistic regression analysis. Receiver operating characteristic (ROC) curve analysis revealed the optimal cut-off points for Siglec-5 was 153.1 ng/ml. After entering Siglec-5, the AUC was 0.99, which was higher than that of confounding risk factors only (AUC = 0.97, P < 0.05). Siglec-5 was expressed in plaques, but not in healthy artery wall in T2DM patients. Elevated plasma Siglec-5 was independently associated with CLI in T2DM. Plasma Siglec-5 levels are implicated as an early diagnostic marker of CLI in T2DM patients and it may become a target for the prevention or treatment of CLI in diabetes.

Critical limb ischemia (CLI) is the most severe manifestation of peripheral artery disease, which is common but rarely diagnosed. Noninvasive biomarkers are urgently required to assist in the diagnosis of CLI. Accumulating evidence indicates that miRNAs play an important role in the development of various diseases. In this study, microarray profiling revealed 11 miRNAs with significantly altered expression in four T2DM patients with CLI compared with that in four sex- and age-matched T2DM patients without CLI. In independent cohorts, qRT-PCR validation confirmed the increased miRNA-4739 level in patients with CLI versus patients without CLI. miRNA-4739 levels increased with FPG and HbA1c (all P < 0.05). After adjusting for the risk factors, miRNA-4739 levels were found to be associated with an increased odds ratio (OR) of T2DM with CLI (OR =12.818, 95% confidence intervals (CI) 1.148 to 143.143, P = 0.038). ROC curve analysis revealed that the area under the curve (AUC) of miR-4739+confounding risk factors was 0.94 (95% CI 0.891 to 0.998, P < 0.001), which was higher than that of confounding risk factors (AUC 0.94 vs. 0.91, 95% CI -0.122 to 0.060, P > 0.05) and of miR-4739 (AUC 0.94 vs. 0.69, 95% CI -0.399 to -0.101, P < 0.001), respectively. We conclude that elevated plasma miRNA-4739 levels are independently associated with CLI in T2DM patients. miRNA-4739 is implicated as a novel diagnostic marker and a potential therapeutic target for CLI in diabetes.

Type 2 diabetes mellitus (T2DM) is a major contributor to peripheral artery disease (PAD), especially in cases that advance to critical limb ischemia (CLI). Accumulating evidence indicates that miRNAs play an important role in the development of PAD and T2DM. Due to the limited value of current diagnostic methods for CLI in T2DM patients, we compared the miRNA expression profiles of Chinese T2DM patients with or without CLI to find out whether distinctive miRNAs could serve as potential diagnostic biomarkers. We statistically identified 7 miRNAs (hsa-miR-200b-3p, hsa-miR-2115-3p, hsa-miR-431-5p, hsa-miR-486-5p, hsa-miR-210-3p, hsa-miR-1264, hsa-miR-323b-5p) which were up-regulated in the CLI group, whereas other 4 miRNAs (hsa-miR-5579-3p, hsa-miR-665, hsa-miR-4285, hsa-miR-500a-3p) were down-regulated. Our validation test suggested a relatively high diagnostic accuracy of serum hsa-miR-323b-5p levels for the detection of CLI in T2DM patients, with a sensitivity of 62.67% and a specificity of 80.65%. The area under the curve (AUC) for miR-323b-5p + confounding risk factors was 0.94 (95% CI: 0.884–0.994, P < 0.001), which was higher than that for miR-323b-5p. Taken together, our results indicate that circulating hsa-miR-323b-5p could be a promising serum biomarker for the diagnosis of critical limb ischemia in type 2 diabetic patients.

Objectives Chronic foot ulceration is a severe complication of diabetes, driving morbidity and mortality. The aim of our study was to identify novel biomarkers of impaired wound healing in diabetic foot ulcers. Methods 109 patients with neuropathic diabetic foot ulcers and 30 burn victims otherwise healthy participated. Antibody-coated glass slide arrays were used to determine the levels of 80 human cytokines in pooled plasma or pooled wound exudate of diabetic foot ulcers with rapidly healing (RH, n = 12) and matched nonhealing (NH, n = 12) patients. Potential biomarkers were confirmed in an independent cohort by enzyme-linked immunosorbent assay (ELISA). Results Protein array profiling identified 27 proteins or 15 proteins significantly altered in protein profiling of pooled plasma or pooled wound exudate of 12 RH patients compared with 12 matched NH patients, respectively. In an independent cohort, quantitative ELISA validation confirmed a decrease in MCP-2 and ENA-78 levels in NH patients versus RH patients or burn victims. After adjusting for the traditional risk factors (sex, age, body mass index, fasting plasma glucose, ulcer area, HbA1C, diabetes duration, hyperlipidemia, and antibiotic therapy), only wound exudate level of ENA-78 remained having a significant association with an increased odds ratio (OR) for wound healing by binary logistic regression analysis (P < 0.05). Conclusion Decreased wound exudate ENA-78 was independently associated with wound healing of patients with diabetic foot. Exudate ENA-78 level is implicated as a novel predictor of wound healing in patients with diabetic foot ulcers.

The study investigated the pharmacodynamism and mechanism of Chinese medicinal formula-Huiru Yizeng Yihao (NO.1 HRYZ) on the model rats of hyperpro-lactinemia and the model rats of hyperplasia of mammary gland (HMG), and studied the internal connection between hyperprolactinemia and HMG.. The hyperprolactinemia rat models were established by injecting metoclopramide dihydrochloride in the back of rats. The model rat of HMG was prepared by injecting estradiol in the thigh muscle of the rats and progesterone consecutively, while the tails of rats were clipped with tongs. Rats were treated with either NO.1 HRYZ or positive control drugs for four weeks. The concentrations of sex hormone in rat serum were examined using ELISA kits, and the morphology of mammary gland tissue in all group rats was observed with microscope. NO.1 HRYZ significantly decreased prolactin (PRL) and increased estradiol (E 2 ), progesterone (P), follicle stimulating hormone (FSH), luteinizing hormone (LH) concentrations of hyperprolactinemia rats. It decreased E 2, PRL, FSH, gonadotropin-releasing hormone (GnRH), 5-hydroxytryptamine (5-HT) and increased P concentrations of HMG rat. It also eliminated hyperplasia of lobules and gland alveolus compared with the model group. Treatment with NO.1 HRYZ could significantly regulate the sex hormone disorder of hyperprolactinemia and HMG rat models, and could eliminate the formation of HMG. Hyperprolactinemia was closely correlated with HMG, and hyperprolactinemia promoted the formation of HMG.

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