Ethics approval and consent to participate
This study was approved by the ethics committee of our University of Medical Sciences, (ethical code: IR.SBMU.MSP.REC.1395.41) and registered in our Registry of Clinical Trials website: (at 14/03/2016) and approved in 19/07/2017, the registration number is: (IRCT2017012932277N1). The study was conducted in the Diabetes Clinic of a teaching Hospital on 22/07/2017 till 18/03/2018. We have explained the study objectives and protocols to the subjects and obtained written informed consent before they participated in the study.
Patients and Study design
In this quasi-experimental study, we chose a total of twenty-seven type-II diabetic patients from our diabetes clinic, considering some inclusion and exclusion criteria. By using sample size formula, we considered the type I error of 5%, type II error of 20%, and standard deviation 5.3 (40.43±5.30) of a previous study (21), thus estimated the sample size of 27 patients.
The study was conducted from July 2017 to March 2018 in compliance with the Helsinki declaration. The understudy patients’ diabetes and their diabetic peripheral neuropathy were diagnosed by a specialized endocrinologist using American Diabetes Association Guideline 2017 (22) and Michigan Neuropathy Screening Instrument (MNSI) (23) respectively. Subjects were randomly screened and selected according to inclusion and exclusion criteria.
The inclusion criteria were as follows: 1- having type-II diabetes for more than 5 years; 2- aged 40-65 years old; 3- having MNSI examination score of more than two; 4- DPNP intensity at least more than 40 millimeters by Visual Analogue Scale (VAS) and 6 months duration; 5- no smoking; 6- no intake of synthetic or herbal drugs for DPNP treatment at least 15 days before the beginning of the study; 7- not using Nitrates containing medicines and Sildenafil; 8- not using synthetic or botanical antioxidants.
The exclusion criteria were: 1- emerging any intolerable adverse drug reaction due to PGB; 2- any neuropathy and pain other than DPNP such as arthritis, gout, surgery, hyperuricemia; 3- developing any acute illnesses such as an infection; 4- having chronic diseases including severe hepatic, renal and cardiovascular disorders; 5- any changes in the kind and dosage of medications, diet, and physical activities during of the study.
Because of human rights, we could not have a control group, and our ethical committee did not approve a group of patients having pain and do not receive any drug for an extended period of two months. Thus the patients received PGB placebo capsules twice/day only for10 days as a washout period and 75mg/Bd PGB capsule in the first week of treatment, followed by 150mg/Bd for seven more weeks. Neither physicians nor the patients were aware of the contents of the capsules. We took the fasting blood samples of the subjects before and after treatment in three-time courses. The first time was before treatment (BT), or at the baseline (after the washout period and before the administration of 75mg PGB. The second sample was taken one month after treatment (OMT), or four weeks later in the middle of the study, and third: eight weeks later or two months after treatment (TMT), at the end of the study.
The Endocrinologist measured the intensity of patients' pain by the VAS method, and we calculated the monthly mean of patients' pain at three times: BT, OMT, and TMT. Many previous studies approved this method for patients' pain measuring. The absence of pain is expressed by zero, and the worst pain is stated 100 millimeters on the ruler. The numbers also represent the pain between these two ranges according to the patient’s feelings (24).
Serum iNOS concentration measurement
The three obtained fasting blood samples of each participant were allowed to clot for 10-15 minutes at room temperature, and then serums were separated by centrifuging (Beckman Avanti J-25; Beckman Coulter, Brea, CA, USA) at 3000 rpm for 10 minutes. Immediately after centrifugation, we stored the supernatant serums at -70°C until iNOS assay. Before the beginning of the analysis, we kept the frozen samples at room temperature (RT) for at least 30 minutes. Then we assessed the concentrations of iNOS in serum samples by applying a commercial enzyme-linked immune sorbent assay (ELISA) kit (ZellBio GmbH, Germany, Cat. No: ZB-10928S-H9648). This kit is based on the Biotin double antibody sandwich technology to assay the human iNOS. The wells of microplates of the package are pre-coated with an anti-iNOS monoclonal antibody. After adding the serum, iNOS of serum adheres to the pre-coated monoclonal antibody. After that, adding anti-iNOS antibody labeled with biotin to combine streptavidin-HRP leading to form an immune complex. Multi-step washing processes removed unbound enzymes. Immune complex plus chromogen solutions created the color, which the related optic density is proportional to the concentration of the iNOS. After adding a stop solution, we measured the optic density of unknown amounts of serum iNOS of patients at 450 nm wavelength using the ELISA reader (BioTek Instruments, Inc., Winooski, VT, USA). Finally, we calculated the iNOS concentrations of serum samples using the standard curve.
Serum NO concentration measurement
Blood samples of each participant were allowed to clot for 10-15 minutes at room temperature, and then serums were separated by centrifuging (Beckman Avanti J-25; Beckman Coulter, Brea, CA, USA) at 3000 rpm for 20 minutes. Then the supernatant serums were stored at -70°C until NO assay via applying a kit based on the Griess colorimetric method (ZellBio GmbH, Germany, Cat. No: ZB-NO-96A).
After gradually defrosting the samples in RT, they were first deproteinized and then assessed by the use of the Griess Method in a 96 flat microplate. We prepared the standard solutions of Nitrate and Nitrite in a serial dilution. We performed the reduction of nitrate to nitrite by adding vanadium-(III)-chloride (8mg/ml). The Griess reagent was made of 50μL sulfanilamide (2%) and 50μL N-(1-Naphthyl) ethylenediamine dihydrochloride (0.1%). After adding Griess reagent to samples, they were incubated at 37ºC for 30 minutes, and after color formation, the absorbance was observed at 540 nm wavelength using the ELISA reader (BioTek Instruments, Inc., Winooski, VT, USA). As a final point, the NO concentrations were calculated using the standard curve.
We analyzed the data using the SPSS19. We used the Kolmogorov-Smirnov test to assess the normality of the data. We used the repeated measure method and paired t-test to detect the effect of intervention and time on iNOS, NO, and DPNP. To assess the correlation of iNOS, NO, and DPNP, we used the Pearson correlation test. We considered the results to be statistically significant at a p-value of less than 0.05.