Identification of an LDLR variant in a Chinese familial hypercholesterolemia and its relation to ROS/NLRP3-Mediated pyroptosis in hepatic cells.

BACKGROUND: Familial hypercholesterolemia (FH) is a common autosomal dominant hereditary disease. Its early diagnosis and intervention significantly improve the patient's quality of life. However, there are few types of research on the FH pathogenic genes in China. METHODS: In this study, we recruited a family diagnosed with FH and used whole exome sequencing (WES) to analyze the proband variants. Intracellular cholesterol level, reactive oxygen species (ROS) level, and the expression of pyroptosis-related genes were detected after overexpression of wild-type or variant LDLR in L02 cells. RESULTS: A heterozygous missense variant predicted to be deleterious to LDLR (c.1879G > A, p.Ala627Thr) was identified in the proband. Mechanistically, intracellular cholesterol level, ROS level, and the expression of pyroptosis-related genes, nucleotide-binding oligomerization domain-like receptor family protein 3 (NLRP3) inflammasome and components (caspase 1, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and NLRP3), gasdermin D (GSDMD), interleukin (IL) -18, IL-1ß was elevated in the variant LDLR group, which was attenuated by inhibition of ROS. CONCLUSIONS: FH is associated with a variant (c.1879G>A, p.Ala627Thr) in the LDLR gene. Regarding the mechanism, the ROS/NLRP3-mediated pyroptosis in hepatic cells may contribute to the pathogenesis of the LDLR variant.

ENA-78 Is a Novel Predictor of Wound Healing in Patients with Diabetic Foot Ulcers.

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.

Circulating MicroRNA-4739 May Be a Potential Biomarker of Critical Limb Ischemia in Patients with 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.

MiR-323b-5p acts as a novel diagnostic biomarker for critical limb ischemia in type 2 diabetic patients.

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.

Cavin-1 regulates caveolae-mediated LDL transcytosis: crosstalk in an AMPK/eNOS/ NF-κB/Sp1 loop.

Caveolae are specialized lipid rafts structure in the cell membrane and critical for regulating endothelial functions, e.g. transcytosis of macromolecules like low density lipoprotein (LDL) etc. Specifically, the organization and functions of caveolae are mediated by structure protein (caveolin-1) and adapter protein (cavin-1). The pathogenic role of caveolin-1 is well studied; nevertheless, mechanisms whereby cavin-1 regulates signaling transduction remain poorly understood. The aim of this study was designed to explore the role of cavin-1 in caveolae-mediated LDL transcytosis across endothelial cells. We reported here that cavin-1 knockdown mediated by small interfering RNA (siRNA) caused a significant decrease of LDL transcytosis. Moreover, cavin-1 knockdown increased the activity of endothelial nitric oxide synthase (eNOS) and the production of nitric oxide (NO). Consequently, an eNOS inhibitor, N-Nitro-L-Arginine Methyl Ester (L-NAME), not only suppressed the activity of specificity protein (Sp1) and nuclear factor kappa B (NF-κB), but also inhibited both activities via activating adenosine 5'-monophosphate- activated protein kinase (AMPK). In conclusion, we proposed an AMPK/eNOS/NF-κB/Sp1 circuit loop was formed to regulate caveolae residing proteins' expression, e.g. LDL receptor (LDLR), caveolin-1, eNOS, thereby to regulate caveolae-mediated LDL transcytosis in endothelial cells.

Siglec-5 is a novel marker of critical limb ischemia in patients with diabetes.

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.

Effect of Chinese medical herbs-Huiru Yizeng Yihao on hyperprolactinemia and hyperplasia of mammary gland in mice.

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 (E2), progesterone (P), follicle stimulating hormone (FSH), luteinizing hormone (LH) concentrations of hyperprolactinemia rats. It decreased E2, 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.

[Cloning, expression and immunologic identification of Eg10 gene of Echinococcus granulosus].

OBJECTIVE: To clone and express Eg10 gene of Echinococcus granulosus, and investigate the immunological characteristic of the recombinant. METHODS: Eg10 gene was subcloned into pET28a vector. The recombinant plasmid was transformed into E. coli BL21 and induced with IPTG. The recombinant protein was purified with His-bind purification kit. Forty-eight mice were randomly divided into 4 groups. Mice in groups A and B were injected with PBS and PBS+Freund's adjuvant (100 microl) as control. Mice in groups C and D were immunized with 10 mg and 50 microg purified Eg10 antigen formulated in Freund's adjuvant, respectively. All the mice received three immunizations at 2-week intervals with the same dose of antigen. Serum samples were collected at pre-immunization and certain time after immunization. The immunological characteristics of recombinant Eg10 was analyzed by Western blotting and ELISA. RESULTS: The recombinant Eg10 protein (Mr 31 000) was expressed in E. coli BL21. The recombinant Eg10 and expression product of PET28a/Eg10 were recognized by sera from mice immunized with recombinant Eg10. ELISA showed that the titer of IgG reached a peak at the 8th week in groups C and D, the level of IgG in sera of groups C or D was higher than that of groups A or B (P < 0.05) at the 2nd, 4th, 6th, 8th, and 10th week. There was no significant difference in the level of IgG between group C and group D (P > 0.05). CONCLUSION: The Eg10 gene has been expressed with immunogenicity.