Luis Miguel Román-Pintos

Diabetic Polyneuropathy in Type 2 Diabetes Mellitus: Inflammation, Oxidative Stress, and Mitochondrial Function

Diabetic polyneuropathy (DPN) is defined as peripheral nerve dysfunction. There are three main alterations involved in the pathologic changes of DPN: inflammation, oxidative stress, and mitochondrial dysfunction. Inflammation induces activation of nuclear factor kappa B, activator protein 1, and mitogen-activated protein kinases. Oxidative stress induced by hyperglycemia is mediated by several identified pathways: polyol, hexosamine, protein kinase C, advanced glycosylation end-products, and glycolysis. In addition, mitochondrial dysfunction accounts for most of the production of reactive oxygen and nitrosative species. These free radicals cause lipid peroxidation, protein modification, and nucleic acid damage, to finally induce axonal degeneration and segmental demyelination. The prevalence of DPN ranges from 2.4% to 78.8% worldwide, depending on the diagnostic method and the population assessed (hospital-based or outpatients). Risk factors include age, male gender, duration of diabetes, uncontrolled glycaemia, height, overweight and obesity, and insulin treatment. Several diagnostic methods have been developed, and composite scores combined with nerve conduction studies are the most reliable to identify early DPN. Treatment should be directed to improve etiologic factors besides reducing symptoms; several approaches have been evaluated to reduce neuropathic impairments and improve nerve conduction, such as oral antidiabetics, statins, and antioxidants (alpha-lipoic acid, ubiquinone, and flavonoids).

The effect of ubiquinone in diabetic polyneuropathy: A randomized double-blind placebo-controlled study

INTRODUCTION Diabetic polyneuropathy aetiology is based on oxidative stress generation due to production of reactive oxygen species. Ubiquinone is reduced to ubiquinol and redistributed into lipoproteins, possibly to protect them from oxidation. AIMS To evaluate the impact of oral ubiquinone in diabetic polyneuropathy, and the role of lipid peroxidation (LPO) and nerve growth factor (NGF-β). METHODS We conducted a double-blind, placebo-controlled clinical trial, patients were randomized to ubiquinone (400 mg) or placebo daily for 12 weeks. Main outcomes were clinical scores, nerve conduction studies, LPO, NGF-β and safety. RESULTS Twenty four patients on experimental group and twenty five on control group met the inclusion criteria (mean age 56 years, 22% male and 78% female, mean evolution of type 2 diabetes mellitus 10.7 years). Significant improvement on experimental vs control group was found in neuropathy symptoms score (from 2.5 ± 0.7 to 1 ± 0.8, p<0.001), neuropathy impairment score (5.5 ± 4 to 3.1 ± 2.6, p<0.001), sural sensory nerve amplitude (13.0 ± 6.1 to 15.8 ± 5.1 μV, p=0.049), peroneal motor nerve conduction velocity (39.7 ± 5.0 to 47.8 ± 4.9 m/s, p=0.047), and ulnar motor nerve conduction velocity (48.8 ± 6.8 to 54.5 ± 6.1m/s, p=0.046). There was a significant reduction of LPO in subjects treated with ubiquinone vs placebo (16.7 ± 8.6 and 23.2 ± 15.8 nmol/mL, respectively) with p<0.05, and NGF-β did not change (control 66.5 ± 26.7 vs. experimental 66.8 ± 28.4 pg/mL, p=0.856). No drug-related adverse reactions were reported. CONCLUSIONS Twelve weeks treatment with ubiquinone improves clinical outcomes and nerve conduction parameters of diabetic polyneuropathy; furthermore, it reduces oxidative stress without significant adverse events.

Effect of rosuvastatin on diabetic polyneuropathy: a randomized, double-blind, placebo-controlled Phase IIa study.

Background Diabetic neuropathy affects 50%–66% of patients with diabetes mellitus. Oxidative stress generates nerve dysfunction by causing segmental demyelinization and axonal degeneration. Antioxidants are considered to be the only etiologic management for diabetic polyneuropathy, and statins such as rosuvastatin increase nitric oxide bioavailability and reduce lipid peroxidation. The aim of this study was to evaluate the antioxidant effect of rosuvastatin in diabetic polyneuropathy. Methods We conducted a randomized, double-blind, placebo-controlled Phase IIa clinical trial in patients with type 2 diabetes and diabetic polyneuropathy (DPN) stage ≥1b. We allocated subjects to two parallel groups (1:1) that received rosuvastatin 20 mg or placebo for 12 weeks. Primary outcomes were neuropathic symptom score, disability score, and nerve conduction studies, and secondary outcomes were glycemic control, lipid and hepatic profile, lipid peroxidation, and nerve growth factor beta (NGF-β) levels. Results Both groups were of similar age and duration since diagnosis of diabetes and DPN. We observed improvement of DPN in the rosuvastatin group from stage 2a (88.2%) to stage 1b (41.2%), improvement of neuropathic symptom score from 4.5±2 to 2.4±1.8, and significant (P=0.001) reductions of peroneal nerve conduction velocity (from 40.8±2.2 to 42.1±1.6 seconds) and lipid peroxidation (from 25.4±2 to 12.2±4.0 nmol/mL), with no significant change in glycemic control or β-NGF. Conclusion The severity, symptoms, and nerve conduction parameters of DPN improved after 12 weeks of treatment with rosuvastatin. These beneficial effects appear to be attributable to reductions in lipid peroxidation and oxidative stress.

Effects of Ezetimibe/Simvastatin and Rosuvastatin on Oxidative Stress in Diabetic Neuropathy: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial

Objective. To evaluate the effects of ezetimibe/simvastatin (EZE/SIMV) and rosuvastatin (ROSUV) on oxidative stress (OS) markers in patients with diabetic polyneuropathy (DPN). Methods. We performed a randomized, double-blind, placebo-controlled phase III clinical trial in adult patients with Type 2 Diabetes Mellitus (T2DM) and DPN, as evaluated by composite scores and nerve conduction studies (NCS). Seventy-four subjects with T2DM were allocated 1 : 1 : 1 to placebo, EZE/SIMV 10/20 mg, or ROSUV 20 mg for 16 weeks. All patients were assessed before and after treatment: primary outcomes were lipid peroxidation (LPO), and nitric oxide (NO) surrogate levels in plasma; secondary outcomes included NCS, neuropathic symptom scores, and metabolic parameters. Data were expressed as mean ± SD or SEM, frequencies, and percentages; we used nonparametric analysis. Results. LPO levels were reduced in both statin arms after 16 weeks of treatment (p < 0.05 versus baseline), without changes in the placebo group. NO levels were not significantly affected by statin treatment, although a trend towards significance concerning increased NO levels was noted in both statin arms. No significant changes were observed for the NCS or composite scores. Discussion. EZE/SIMV and ROSUV are superior to placebo in reducing LPO in subjects with T2DM suffering from polyneuropathy. This trial is registered with NCT02129231.

The effect of ubiquinone and combined antioxidant therapy on oxidative stress markers in non-proliferative diabetic retinopathy: A phase IIa, randomized, double-blind, and placebo-controlled study.

Objective To evaluate the effect of ubiquinone (Coenzyme Q10) and combined antioxidant therapy (CAT) on oxidative stress markers in non-proliferative diabetic retinopathy (NPDR) under clinical management. Study design In a randomized, double-blind, phase IIa, placebo-controlled, clinical trial, three study groups were formed and administered medications as follows: Group 1, Coenzyme Q10; Group 2, CAT; and Group 3, placebo. Methods Serum levels of the products of lipid peroxidation (LPO) and nitrites/nitrates, as markers of oxidative/nitrosative stress, were measured. As antioxidants, the total antioxidant capacity (TAC), catalase activity, and glutathione peroxidase (GPx) activity were measured. Results Baseline serum levels of LPO and nitrites/nitrates were significantly elevated in the three groups vs. healthy group (P < 0.0001), while final levels in the Coenzyme Q10 and CAT groups were decreased vs. normal levels (P < 0.0001). The baseline TAC was consumed in the three groups (P < 0.0001), while final results in the Coenzyme Q10 and CAT groups improved (P < 0.0001). Baseline catalase activity was increased in all groups vs. normal values (P < 0.001), while final levels in the Coenzyme Q10 (P < 0.001) and CAT groups (P < 0.0001) were decreased. GPx behaved similarly to catalase and improved in the final results (P < 0.0001). Discussion Adjunctive antioxidant treatment for 6 months was effective and safe for improving the oxidative stress in NPDR.

The antioxidant effect of ubiquinone and combined therapy on mitochondrial function in blood cells in non-proliferative diabetic retinopathy: A randomized, double-blind, phase IIa, placebo-controlled study.

Objectives: To evaluate the effect of ubiquinone and combined antioxidant therapy on mitochondrial function in non-proliferative diabetic retinopathy (NPDR) in a randomized, double-blind, phase IIa, placebo-controlled, clinical trial. Three groups of 20 patients were formed: Group 1, ubiquinone; Group 2, combined therapy; and Group 3, placebo (one daily dose for 6 months). Methods: Fluidity of the submitochondrial membrane in platelets was determined by examining intensity of fluorescence between the monomer (Im) and excimer (Ie). Hydrolytic activity of the mitochondrial F0F1-ATPase was evaluated with the spectrophotometric method. Results: Normal, baseline submitochondrial membrane fluidity, 0.24 ± 0.01 Ie/Im, was significantly diminished in the three study groups vs. normal values (P < 0.0001); placebo, 0.14 ± 0.01 Ie/Im; ubiquinone, 0.14 ± 0.01 Ie/Im; and combined therapy, 0.13 ± 0.00 Ie/Im. Afterward, it increased significantly (P < 0.0001), the ubiquinone group 0.22 ± 0.01 Ie/Im, combined therapy group, 0.19 ± 0.01 Ie/Im; with no changes the placebo group. Baseline hydrolytic activity of the F0F1-ATPase enzyme increased in the three study groups vs. normal values (184.50 ± 7.84 nmol PO4), placebo, 304.12 ± 22.83 nmol PO4 (P < 0.002); ubiquinone, 312.41 ± 25.63 nmol PO4 (P < 0.009); and combined therapy, 371.28 ± 33.50 nmol PO4 (P < 0.002). Afterward, a significant decrease the enzymatic activity: ubiquinone, 213.25 ± 14.19 nmol PO4 (P < 0.001); and combined therapy, 225.55 ± 14.48 nmol PO4 (P < 0.0001). Discussion: Mitochondrial dysfunction significantly improved in groups of NPDR patients treated with antioxidants.

Toll-Like Receptors in Secondary Obstructive Cholangiopathy

Secondary obstructive cholangiopathy is characterized by intra- or extrahepatic bile tract obstruction. Liver inflammation and structural alterations develop due to progressive bile stagnation. Most frequent etiologies are biliary atresia in children, and hepatolithiasis, postcholecystectomy bile duct injury, and biliary primary cirrhosis in adults, which causes chronic biliary cholangitis. Bile ectasia predisposes to multiple pathogens: viral infections in biliary atresia; Gram-positive and/or Gram-negative bacteria cholangitis found in hepatolithiasis and postcholecystectomy bile duct injury. Transmembrane toll-like receptors (TLRs) are activated by virus, bacteria, fungi, and parasite stimuli. Even though TLR-2 and TLR-4 are the most studied receptors related to liver infectious diseases, other TLRs play an important role in response to microorganism damage. Acquired immune response is not vertically transmitted and reflects the infectious diseases history of individuals; in contrast, innate immunity is based on antigen recognition by specific receptors designated as pattern recognition receptors and is transmitted vertically through the germ cells. Understanding the mechanisms for bile duct inflammation is essential for the future development of therapeutic alternatives in order to avoid immune-mediated destruction on secondary obstructive cholangiopathy. The role of TLRs in biliary atresia, hepatolithiasis, bile duct injury, and primary biliary cirrhosis is described in this paper.

Effect of statins on oxidative DNA damage in diabetic polyneuropathy

Oxidative stress induces nerve damage in type 2 diabetes mellitus and leads to diabetic polyneuropathy (DPN) and can affect the DNA and antioxidant status. Statins have pleiotropic, protective effects on the peripheral nerves of patients with diabetes. The aim of this study was to determine the effects of ezetimibe/simvastatin and rosuvastatin on DNA damage in patients with DPN. This randomized, double-blind, placebo-controlled, clinical trial comprised outpatients from Guadalajara, Mexico. The inclusion criteria were either gender, age 35–80 years, type 2 diabetes, glycated hemoglobin ≤10%, diabetic polyneuropathy stage 1/2, and signed informed consent. Patients who were taking antioxidant therapy or statins, had hypersensitivity to drugs, experienced organ failure, were pregnant or breastfeeding, or had other types of neuropathy were excluded. We assigned patients to placebo, ezetimibe/simvastatin 10/20 mg, or rosuvastatin 20 mg, and the primary outcomes were 8-hydroxy-2′-deoxyguanosine (8-OHdG) for DNA damage, 8-oxoguanine-DNA-N-glycosilase (hOGG1) for DNA repair, and superoxide dismutase (SOD). Seventy-four patients were recruited. Nine patients were included as negative controls. There were no differences in 8-OHdG between the healthy subjects (4.68 [3.53–6.38] ng/mL) and the DPN patients (4.51 [1.22–9.84] ng/mL), whereas the hOGG1 level was 0.39 (0.37–0.42) ng/mL in the healthy subjects and 0.41 (0.38–0.54) ng/mL in patients with DPN at baseline (p = 0.01). SOD decreased significantly in patients with DPN (5.35 [0.01–17.90] U/mL) compared with the healthy subjects (9.81 [8.66–12.61] U/mL) at baseline (p < 0.001). No significant changes in DNA biomarkers were observed in any group between baseline and final levels. We noted a rise in hOGG1 in patients with DPN, without modifications after treatment. There was a slight, albeit insignificant, increase in SOD in patients who were on statins.

Ubiquinone, Ezetimibe/Simvastatin and Rosuvastatin Effects on Mitochondrial Function in Diabetic Polyneuropathy

Diabetic polyneuropathy (DPN) pathophysiologic findings include loss of multifocal and focal nerve fibers secondary to axonal degeneration and segmental demyelization due to oxidative stress and mitochondrial dysfunction induced by chronic hypergly‐ caemia. Aim: To evaluate the effect of ubiquinone, ezetimibe/simvastatin and rosuvastatin on mitochondrial function in patients with diabetic polyneuropathy. Methods: A randomized, double‐blinded, placebo‐controlled clinical trial was performed in patients with type 2 Diabetes Mellitus (T2DM) who had DPN, glycated haemoglobin (HbA1c) <12% (108 mmol/mol), previous exclusion of other type of neuropathy. Ninety‐eight persons with T2DM were enrolled allocated 1:1:1:1 to either placebo, ubiquinone 400 mg, ezetimibe/simvastatin 10/20 mg or rosuvastatin 20 mg for 16 weeks. Primary outcomes were F0F1‐ATP hydrolysis, erythrocyte and sub‐mito‐ chondrial platelet membrane fluidity. Results were expressed as mean ± SD or SEM and percentages. Results: F0F1‐ATP hydrolysis levels in healthy controls were 236.80±118.42 nmol/PO4; all patients with T2DM exhibited an increase, but only rosuvastatin demonstrated an improvement with baseline 463.37±47.07 nmol/PO4 vs. after 340.61±37.80 nmol/PO4 treatment (p<0.05). Plasma and sub‐mitochondrial membrane fluidity did not experi‐ ence any significant changes.

Toll-Like Receptor-1 and Receptor-2 and Beta-Defensin in Postcholecystectomy Bile Duct Injury

Postcholecystectomy bile duct injuries (BDI) produce hepatic cholestasis and cause infection of the biliary tract. The biliary cells participate in secreting cytokines and in expression of immune response receptors. Toll-like receptors (TLRs) conduct signalling and activate the innate and adaptive inflammatory response. The objective was to determine the serum levels of TLR-2 and the expression of TLR-1 and TLR-2 and β-defensin in liver biopsies of postcholecystectomy BDI patients. A transverse, analytical study with 2 groups was done. One group included healthy volunteers (control group) and other included 25 postcholecystectomy BDI patients with complete biliary obstruction. Using the Enzyme-linked Immunosorbent Assay (ELISA) technique, serum levels of TLR-2 were determined, and with immunofluorescence the morphologic analysis of TLR-1 and TLR-2 and β-defensin in liver biopsies of postcholecystectomy BDI patients was performed. The average TLR-2 serum level in the control group was 0.0 pg/mL and in the BDI group, 0.023 ± 0.0045 pg/mL (P < 0.0001, bilateral Mann Whitney U). Immunofluorescence was used to determine the expression in liver biopsies, blood vessels, bile ducts, and hepatic parenchyma where 12 hepatic biopsies were positive for TLR-1 with average of 3213057.74 ± 1071019.25 μm2; and 7 biopsies were positive for β-defensin with an average of 730364.33 ± 210838.02 μm2; and 6 biopsies positive for TLR-2, obtaining an average of 3354364.24 ± 838591.06 μm2. In conclusion, TLR-1 and TLR-2 and β-defensin play an important role in the innate antimicrobial defense of the hepatobiliary system.