Decreased glucagon in diabetic peripheral neuropathy patients with long duration type 2 diabetes.

OBJECTIVE: The aim of this study was to investigate the association of fasting C-peptide and glucagon with diabetic peripheral neuropathy (DPN) in patients with type 2 diabetes (T2DM). METHODS: A comprehensive evaluation was conducted on 797 patients with T2DM to assess the various risk factors affecting DPN. The subjects were categorized into short duration and long duration group according to the duration of diabetes with a threshold of 10 years. Logistic regression analysis was employed to examine the association between DPN and islet function, as well as other parameters. Receiver operating characteristic curve analysis was performed to evaluate the predictive capability of glucagon. RESULTS: The fasting C-peptide levels were significantly lower in the DPN patients with short duration of diabetes, but lost significance in the long duration group. Conversely, a decreased level of glucagon was only observed in DPN patients with long duration of diabetes. For the group with long duration of diabetes, glucagon was the sole risk factor associated with DPN. The receiver operating characteristic curve analysis revealed that glucagon in the long duration group exhibited a moderate area under the curve of 0.706. CONCLUSIONS: The serum glucagon levels in T2DM patients with DPN exhibited bidirectional changes based on the duration of diabetes. Decreased glucagon was associated with DPN in T2DM patients with long duration of diabetes.

Ultrasound Intensify the Flavonoid Production of the Willow Bracket Mushroom, Phellinus igniarius (Agaricomycetes), Fermentation Mycelia.

This research aimed to use a novel and effective ultrasound (US) approach for obtaining high bio-compound production, hence proposing strategies for boosting active ingredient biosynthesis. Furthermore, the US promotes several physiological effects on the relevant organelles in the cell, morphological effects on the structure of Phellinus igniarius mycelium, and increases the transfer of nutrients and metabolites. One suitable US condition for flavonoid fermentation was determined as once per day for 7-9 days at a frequency 22 + 40 kHz, power density 120 W/L, treated 10 min, treatment off time 7 s. The flavonoid content and production increased about 47.51% and 101.81%, respectively, compared with the untreated fermentation (P < 0.05). SEM showed that sonication changes the morphology and structure of Ph. igniarius mycelium; TEM reveals the ultrasonic treatment causes organelle aggregation. The ultrasound could affect the metabolism of the biosynthesis of the active ingredients.

Thalidomide Alleviates Apoptosis, Oxidative Damage and Inflammation Induced by Pemphigus Vulgaris IgG in HaCat Cells and Neonatal Mice Through MyD88.

Purpose: Thalidomide (Tha) can be used as a selective treatment for mild pemphigus vulgaris (PV). However, the specific mechanism of action remains unclear. Patients and Methods: PV IgG extracted from patients' serum was cocultured with HaCaT cells to construct a PV cell model, and different concentrations of Tha were used to screen the drug effect. The expression level of MYD88 was assessed in skin lesions of PV patients. Intracellular Ca2+ concentration, reactive oxygen species level, DSG3, PG, MYD88, apoptosis-related proteins (Caspase-3, Bcl-2, and Bax), NF-κB pathway-related proteins (IκBα, p-IκBα, p50, and p65), NLRP3, IFN-γ, TNF-α, IL-6, and IL-8 levels were measured. PV IgG was subcutaneously injected into C57BL/6 neonatal mice to construct the animal model. Immunofluorescence was used to detect IgG deposition in the mouse epidermis, whereas immunohistochemistry and TUNEL methods were used to detect the expression of MYD88 and NLRP3 as well as cell apoptosis level in the mouse epidermis. Results: Tha reversed the decrease in Dsg3 and PG caused by PV IgG. The expression of MyD88 increased in the patients' skin, PV cell model, and PV mouse model. The increase in MyD88 expression level in PV cell models and PV newborn mouse models was inhibited by Tha. Overexpression of MyD88 induced a decrease in the expression levels of Dsg3 and PG in Hacat cells. Overexpression of MyD88 inhibited Tha effects on Dsg3 and PG expressions and blocked Tha effects on Ca2+, apoptosis, Bax, Bcl-2, and Caspase-3 expressions, oxidative damage, and inflammatory response in HaCat cells. Tha alleviated acantholysis induced by PV IgG in model mice. Conclusion: Through MYD88, Tha attenuated apoptosis of HaCat cells, modulated NF-κB to hamper the oxidative damage and inflammatory response in the PV cell models, and alleviated acantholysis, IgG deposition, and epidermal cell apoptosis induced by PV IgG in model mice.

Efficacy of long-term repeated rituximab treatment in refractory phospholipase A2 receptor-antibody-related membranous nephropathy.

Aims: To evaluate the efficacy of long-term repeated rituximab treatment in refractory PLA2R-Ab-related membranous nephropathy (MN). Materials & methods: Rituximab was administered at a single dose of 375 mg/m2 and repeated if peripheral B-cell levels were >5/ul in 46 patients with refractory PLA2R-Ab-related MN. Results: The median frequency of rituximab treatment was 3 (IQR 2.0-4.0). A total of 32 (32/46) patients achieved remission (completed remission [CR] or partial remission [PR]) over a median time of 17.0 months, and 10 patients eventually progressed to CR. The proportion of serum PLA2R-Ab depletion was 73.91% (34/46) over a median time of 9 months. Antibody depletion preceded proteinuria remission. Conclusions: Long-term repeated rituximab treatment achieved high kidney and immunological response rates in refractory PLA2R-Ab related MN, and antibody depletion was a prerequisite for proteinuria remission.

Membranous nephropathy (MN) is the leading cause of proteinuria in adults and is mainly manifested as edema. Phospholipase 2 receptor (PLA2R) antibody is detected in 70­80% of patients with MN. Its main treatments are immunosuppressive therapies. The efficacy of traditional immunosuppressant drugs including steroids and alkylating and calcineurin inhibitors in reducing proteinuria have been confirmed; however, several adverse events such as diabetes mellitus, infections, cancer and kidney injury have been reported. Rituximab, a monoclonal antibody against CD20 on B cells, has been verified to be effective, safer and better tolerated in MN. However, the optimal rituximab regimen for MN has not yet been established. Here, we use B-cell-driven long-term repeated rituximab regimen and determine its exciting efficacy for refractory MN.

The Early Prediction of Common Disorders in Dairy Cows Monitored by Automatic Systems with Machine Learning Algorithms.

We use multidimensional data from automated monitoring systems and milking systems to predict disorders of dairy cows by employing eight machine learning algorithms. The data included the season, days in milking, parity, age at the time of disorders, milk yield (kg/day), activity (unitless), six variables related to rumination time, and two variables related to the electrical conductivity of milk. We analyze 131 sick cows and 149 healthy cows with identical lactation days and parity; all data are collected on the same day, which corresponds to the diagnosis day for disordered cows. For disordered cows, each variable, except the ratio of rumination time from daytime to nighttime, displays a decreasing/increasing trend from d-7 or d-3 to d0 and/or d-1, with the d0, d-1, or d-2 values reaching the minimum or maximum. The test data sensitivity for three algorithms exceeded 80%, and the accuracies of the eight algorithms ranged from 65.08% to 84.21%. The area under the curve (AUC) of the three algorithms was >80%. Overall, Rpart best predicts the disorders with an accuracy, precision, and AUC of 81.58%, 92.86%, and 0.908, respectively. The machine learning algorithms may be an appropriate and powerful decision support and monitoring tool to detect herds with common health disorders.

Akt-mediated platelet apoptosis and its therapeutic implications in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet count which can cause fatal hemorrhage. ITP patients with antiplatelet glycoprotein (GP) Ib-IX autoantibodies appear refractory to conventional treatments, and the mechanism remains elusive. Here we show that the platelets undergo apoptosis in ITP patients with anti-GPIbα autoantibodies. Consistent with these findings, the anti-GPIbα monoclonal antibodies AN51 and SZ2 induce platelet apoptosis in vitro. We demonstrate that anti-GPIbα antibody binding activates Akt, which elicits platelet apoptosis through activation of phosphodiesterase (PDE3A) and PDE3A-mediated PKA inhibition. Genetic ablation or chemical inhibition of Akt or blocking of Akt signaling abolishes anti-GPIbα antibody-induced platelet apoptosis. We further demonstrate that the antibody-bound platelets are removed in vivo through an apoptosis-dependent manner. Phosphatidylserine (PS) exposure on apoptotic platelets results in phagocytosis of platelets by macrophages in the liver. Notably, inhibition or genetic ablation of Akt or Akt-regulated apoptotic signaling or blockage of PS exposure protects the platelets from clearance. Therefore, our findings reveal pathogenic mechanisms of ITP with anti-GPIbα autoantibodies and, more importantly, suggest therapeutic strategies for thrombocytopenia caused by autoantibodies or other pathogenic factors.

Efficacy analysis of self-help position therapy after holmium laser lithotripsy via flexible ureteroscopy.

BACKGROUND: To observe the efficacy of self-help position therapy (SHPT) after holmium laser lithotripsy via flexible ureteroscopy (FURS). METHODS: From January 2010 to November 2015, 736 nephrolithiasis patients who had received FURS lithotripsy were analyzed retrospectively. In position group, 220 cases accepted SHPT after lithotripsies, and 428 cases as control, coming from another independent inpatient area in the same center. The stone-free status (SFS) between two groups were compared at the 2nd, 4th and 12th week ends by X-ray examinations. RESULTS: The preoperative incidence of hydronephrosis (25.9% vs. 18.0%, p = 0.018) or lower calyceal seeper (33.6% vs. 24.3%, p = 0.012) and the proportion of patients with > 2.0 cm stones (33.6% vs. 24.3%, p = 0.003) were all significantly higher in position group than in control group. There were no substantial difference between two groups in age, BMI, gender and medical histories. In postoperative followup, the incidence of hydronephrosis in position group was significantly lower than in control group (9.5% vs. 15.7%, p = 0.032) after removing double-J stents. In position group, the SFS of the 2nd week end (60.9% vs. 47.2%, p = 0.001), the 4th week end (74.1% vs. 62.8%, p = 0.004) and the 12th week end (86.9% vs. 79.4%, p = 0.021) were all significantly higher than those in control group. CONCLUSIONS: SHPT after holmium laser lithotripsy via FURS may increase postoperative SFS, accelerate stone fragment clearance, and decrease the incidence of hydronephrosis after removal of double-J stents. The therapy does not require professional assistance and is economical, simple, and effective.

Effects of fluoride on morphology, growth, development, and thyroid hormone of Chinese toad (Bufo gargarizans) embryos.

Excessive fluoride in natural water ecosystem has the potential to detrimentally affect amphibians, but little is known of such effects or underlying mechanisms in Bufo gargarizans embryos. In the present study, the effects of fluoride exposure on B. gargarizans embryos were investigated. First, fluoride teratogenic experiment showed that the 9 days EC50 of fluoride on B. gargarizans embryos was 177.62 mg/L. Then, we studied the sublethal effects of fluoride on B. gargarizans embryos at control, 0.7, 4.1, 19.6, 41.9, and 62.7 mg/L fluoride concentration. Malformation, growth, and development of embryos were monitored, and type 2 and 3 iodothyronine deiodinase (Dio2 and Dio3), thyroid hormone receptors (TRα and TRß) mRNA levels were measured. Our results showed the morphological malformations, such as tail curvature (lordosis), edema, cuticularized ciliated cells, and hyperplasia were occurred during fluoride exposure. Growth and development were all inhibited at 19.5, 41.9, and 62.7 mg/L fluoride-treated groups after 9 days' exposure. According to real-time PCR results, exposure to fluoride upregulated Dio3 and TRß mRNA expression and downregulated Dio2 and TRα mRNA level. All above indicated that excessive fluoride could induce morphology malformations, inhibit embryonic growth and development, and disrupt the normal function of maternal thyroid hormone in B. gargarizans embryos. Environ. Mol. Mutagen. 59:123-133, 2018. © 2017 Wiley Periodicals, Inc.

Protein kinase A determines platelet life span and survival by regulating apoptosis.

Apoptosis delimits platelet life span in the circulation and leads to storage lesion, which severely limits the shelf life of stored platelets. Moreover, accumulating evidence indicates that platelet apoptosis provoked by various pathological stimuli results in thrombocytopenia in many common diseases. However, little is known about how platelet apoptosis is initiated or regulated. Here, we show that PKA activity is markedly reduced in platelets aged in vitro, stored platelets, and platelets from patients with immune thrombocytopenia (ITP), diabetes, and bacterial infections. Inhibition or genetic ablation of PKA provoked intrinsic programmed platelet apoptosis in vitro and rapid platelet clearance in vivo. PKA inhibition resulted in dephosphorylation of the proapoptotic protein BAD at Ser155, resulting in sequestration of prosurvival protein BCL-XL in mitochondria and subsequent apoptosis. Notably, PKA activation protected platelets from apoptosis induced by storage or pathological stimuli and elevated peripheral platelet levels in normal mice and in a murine model of ITP. Therefore, these findings identify PKA as a homeostatic regulator of platelet apoptosis that determines platelet life span and survival. Furthermore, these results suggest that regulation of PKA activity represents a promising strategy for extending platelet shelf life and has profound implications for the treatment of platelet number-related diseases and disorders.

Receptor-interacting protein kinase 3 promotes platelet activation and thrombosis.

Previous studies have shown that receptor-interacting protein kinase 3 (RIP3) is involved in many important biological processes, including necroptosis, apoptosis, and inflammation. Here we show that RIP3 plays a critical role in regulating platelet functions and in vivo thrombosis and hemostasis. Tail bleeding times were significantly longer in RIP3-knockout (RIP3-/-) mice compared with their wild-type (WT) littermates. In an in vivo model of arteriole thrombosis, mice lacking RIP3 exhibited prolonged occlusion times. WT mice repopulated with RIP3-/- bone marrow-derived cells had longer occlusion times than RIP3-/- mice repopulated with WT bone marrow-derived cells, suggesting a role for RIP3-deficient platelets in arterial thrombosis. Consistent with these findings, we observed that RIP3 was expressed in both human and mice platelets. Deletion of RIP3 in mouse platelets caused a marked defect in aggregation and attenuated dense granule secretion in response to low doses of thrombin or a thromboxane A2 analog, U46619. Phosphorylation of Akt induced by U46619 or thrombin was diminished in RIP3-/- platelets. Moreover, RIP3 interacted with Gα13 Platelet spreading on fibrinogen and clot retraction were impaired in the absence of RIP3. RIP3 inhibitor dose-dependently inhibited platelet aggregation in vitro and prevented arterial thrombus formation in vivo. These data demonstrate a role for RIP3 in promoting in vivo thrombosis and hemostasis by amplifying platelet activation. RIP3 may represent a novel promising therapeutic target for thrombotic diseases.

Ethanol Induces Platelet Apoptosis.

BACKGROUND: Alcohol abuse incurs severe medical conditions, such as thrombocytopenia and hemorrhage, but the pathogenesis is not totally understood. Alcohol has been reported to induce apoptosis in eukaryotic cells, such as hepatocyte, nerve cell, corneal fibroblasts. However, it is still unclear whether alcohol induces platelet apoptosis. METHODS: Washed human platelets were pretreated with ethanol (EtOH), and apoptotic events and platelet function were detected. In in vivo experiments, C57BL/6J mice were given EtOH by gavage. Platelet counts, tail bleeding time, and the stomach were examined. RESULTS: EtOH dose dependently induces depolarization of mitochondrial inner transmembrane potential, up-regulation of Bax, down-regulation of Bcl-2, and caspase-3 activation. EtOH does not induce surface expression of P-selectin or PAC-1 binding, whereas significantly reduces collagen-, thrombin-, and ADP-induced platelet aggregation. Moreover, EtOH induces c-Jun NH2-terminal kinase activation. In an in vivo mouse model of the acute alcoholism, EtOH significantly reduces the number of circulating platelets, prolongs the tail bleeding time, and causes gastric mucosa hemorrhage. CONCLUSIONS: These data demonstrate that EtOH induces mitochondria-mediated intrinsic platelet apoptosis, results in the reduction of the number of circulating platelets, and impairs in vivo hemostasis. These findings reveal the possible pathogenesis of hemorrhagic symptoms in patients experiencing acute alcohol intoxication.

Lovastatin induces platelet apoptosis.

Statins are widely used in the prevention of atherosclerosis and treatment of coronary artery disease because of pleiotropic effects on thrombosis. Thrombocytopenia and hemorrhage occurred in some statin-treated patients, but the reason remains unclear. In the current study, we show that lovastatin dose-dependently induces depolarization of mitochondrial inner transmembrane potential, leading to up-regulation of Bak, down-regulation of Bcl-XL, and activation of caspase-3/8/9. Lovastatin treatment did not increase the surface expression of P-selectin or PAC-1 binding but led to strongly reduced collagen- and thrombin-induced platelet aggregation. The integrin αIIbß3 antagonist, RGDS, inhibited lovastatin-induced apoptosis in both human platelets and Chinese hamster ovary (CHO) cells stably expressing integrin αIIbß3. The number of circulating platelets in mice was significantly reduced after intraperitoneal injections with lovastatin. Taken together, these data indicate that lovastatin induced caspase-dependent platelet apoptosis. Lovastatin does not incur platelet activation, whereas impairs platelet function and reduces circulating platelets in vivo, suggesting the possible pathogenesis of thrombocytopenia and hemorrhage in patients treated with statins.

Staurosporine Induces Platelet Apoptosis Through p38 Mitogen-Activated Protein Kinase Signaling Pathway.

BACKGROUND: Staurosporine (STS), a microbial alkaloid and potent PKC inhibitor, has become one of the most promising anti-cancer drugs. STS effectively induces apoptosis in many nucleated cells; however, it is still unclear whether STS induces apoptosis in enucleated platelets. METHODS: Apoptotic events in platelets treated with STS were assessed by flow cytometry or western blotting. RESULTS: STS induced depolarization of mitochondrial inner transmembrane potential (ΔΨm), up-regulation of Bax and Bak, phosphatidylserine (PS) exposure, release of mitochondrial cytochrome c, and activation of caspase-8 and caspase-9 in human platelets. Furthermore, STS stimulation induced phosphorylation of p38 mitogen-activated protein kinase (MAPK). Inhibition of p38 MAPK activation significantly reduced ΔΨm depolarization and PS exposure in platelets stimulated with STS. CONCLUSIONS: These data indicate that STS induces platelet apoptosis via the p38 MAPK signaling pathway. These findings suggest that platelet apoptosis-related hemorrhage should be noticed in STS and its derivatives in clinical tests.

Glycoprotein Ibα clustering induces macrophage-mediated platelet clearance in the liver.

Many immune thrombocytopenia (ITP) patients, particularly patients with anti-glycoprotein (GP) Ib-IX autoantibodies, do not respond to the conventional treatments such as splenectomy. However, the underlying mechanism remains unclear. Here we found that anti-GPIbα N-terminus antibody AN51, but not other anti-GPIbα antibodies (AK2, HIP1, VM16d, or WM23), induced GPIbα clustering that led to integrin αIIbß3-dependent platelet aggregation. After intravenous injection, AN51 dose-dependently induced thrombocytopenia in guinea pigs, and the platelets were mainly removed by macrophages in the liver. N-acetyl-D-glucosamine, previously shown to inhibit integrin αMß2-mediated phagocytosis of refrigerated platelets, dose-dependently inhibited AN51-induced platelet clearance. Furthermore, AN51 but not VM16d, induced rapid platelet clearance in the liver of cynomolgus macaques. Five of 22 chronic ITP patients had anti-GPIbα autoantibodies, and the autoantibodies from four of the five patients competed with AN51 for binding to platelets. These data indicate that GPIbα clustering induced by anti-GPIbα N-terminus antibody causes integrin αIIbß3-dependent platelet aggregation, phagocytosis, and rapid platelet clearance in the liver. Our findings reveal a novel Fc-independent mechanism underlying the pathogenesis of ITP, and suggest new therapeutic strategies for ITP patients with anti-GPIbα autoantibodies.

Carmustine induces platelet apoptosis.

Carmustine is one of the alkylating chemotherapeutic agents, which are used to treat various types of cancers, such as brain tumors, Hodgkins and non-Hodgkins lymphoma and multiple myeloma. However, carmustine has the side effect of thrombocytopenia, and the mechanism is not completely understood. In this study, we show that carmustine dose-dependently induced depolarization of mitochondrial inner transmembrane potential (ΔΨm), up-regulation of Bax, down-regulation of Bcl-2 and caspase-3 activation. Carmustine did not induce surface expression of P-selectin or PAC-1 binding, whereas, obviously reduced collagen and thrombin-induced platelet aggregation. Dicumarol, c-Jun NH2-terminal kinase-specific inhibitor, reduced carmustine-induced ΔΨm depolarization in platelets. The numbers of circulating platelets were reduced, and the tail bleeding time was significantly increased in mice that were injected with carmustine. Taken together, these data indicate that carmustine induced platelet apoptosis, suggesting the possible pathogenesis of thrombocytopenia in patients treated with carmustine.

[Protein kinase C activation induces platelet apoptosis].

Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelets, or in the development of some platelet-related diseases. However, up to now, a little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase C (PKC) is highly expressed in platelets and plays central roles in regulating platelet functions. Although there is evidence indicating that PKC is involved in the regulation of apoptosis of nucleated cells, it is still unclear whether PKC plays a role in platelet apoptosis. The aim of this study was to investigate the role of PKC in platelet apoptosis. The effects of PKC on mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) exposure, and caspase-3 activation of platelets were analyzed by flow cytometry and Western blot. The results showed that the ΔΨm depolarization in platelets was induced by PKC activator in time-dependent manner, and the caspase-3 activation in platelets was induced by PKC in concentration-dependent manner. However, the platelets incubated with PKC inhibitor did not results in ΔΨm depolarization and PS exposure. It is concluded that the PKC activation induces platelet apoptosis through influencing the mitochondrial functions and activating caspase 3. The finds suggest a novel mechanism for PKC in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis.

Aspirin Induces platelet apoptosis.

Aspirin is widely used in the treatment of a number of clinical conditions. Although aspirin is being thought to be a relatively "safe" medicine, it also has some side effects, particularly the risk of bleeding which may be severe and lead to death. The mechanisms, however, are not totally understood. It has been reported recently that aspirin induces apoptosis in many cell types. Thus, the aim of the current study is to explore whether aspirin induces platelet apoptosis. The data show that mitochondrial transmembrane potential (ΔΨm) depolarizations and phosphatidylserine (PS) exposures were dose-dependently induced by aspirin in platelets. To further confirm that aspirin incurs platelet apoptosis, caspase-3 activity was measured in platelets, and the result indicated that aspirin induced caspase-3 activation. Furthermore, the mean volume of platelets incubated with aspirin was obviously reduced. Caspase inhibitor z-VAD-fmk inhibited aspirin induced apoptotic platelet shrinkage and ΔΨm depolarization, but had no effect on PS exposure. In addition, platelets incubated with cyclooxygenase inhibitor indomethacin did not incur ΔΨm depolarazation and PS exposure. Taken together, the data indicate that aspirin induces platelet apoptosis via caspase-3 activation.