Introduction: Diabetes is a chronic metabolic disease and a major health problem that is on the rise, especially in developing countries. Overall, the number of patients with diabetes in the world is more than 250 million and is projected to increase by 350 million in 2020 and more than 438 million in 2030. The chronic nature of diabetes has a great impact on the patient's body-mind and individual and social functioning, and therefore the study of various aspects of health in these patients is of particular importance. (Mirsifi et al., 2018). As mentioned, chronic diabetes occurs when the body is unable to produce enough insulin or to use insulin effectively, which can be due to genetic factors and environmental factors. (Harris and Zimmet, 1997).
In 2011, there were 366 million people with diabetes worldwide, half of whom are still unaware of their disease. It is also predicted that by 2030 the number of people with diabetes worldwide will reach 552 million. The prevalence of type 2 diabetes is becoming a major problem for human health because the figures for the statistics provided in the coming decade are doubling, and as a result, diabetes in adults will become a problem. Weight gain, sedentary lifestyle, and diet are some of the factors that contribute to type 2 diabetes. (Sally et al., 2004). Type 2 diabetes (T2DM) is the most common form of diabetes and causes impairment in insulin secretion and insulin resistance (Deferonzo et al., 2007).
As mentioned above, chronic diabetes occurs when the body is unable to produce enough insulin or use insulin effectively, which can be due to genetic backgrounds and environmental factors. In 2011, around the world, 366 million people had diabetes, half of whom are not yet aware of their disease. It is also predicted that by 2030 the number of people with diabetes worldwide will reach 552 million. The prevalence of type 2 diabetes is becoming a major problem facing human health as the amounts of statistics presented in the decade ahead are doubling, resulting in diabetes in adults becoming a problem. Weight gain, the prevalence of living, and low-stimulation life, and diet are among the factors in the incidence of type 2 diabetes. The prevalence of this patient in European countries is 7% of the total population, which is 5-7% of the total population in the world. This estimate cannot be so real because in many cases it can be said that 50% of people with diabetes are unknown. Studies show that the rate of diabetes in urban areas is higher than in rural areas.
The overall goal in managing type 2 diabetes is to control blood glucose and reduce the risk of long-term complications. Numerous studies have shown that modern management can limit, delay, and even prevent the long-term effects of diabetes by tightly controlling blood sugar levels.
Managing blood sugar in type 2 diabetes has become a complex and somewhat controversial process because different drugs have been suggested to do so, but the increase in available drugs has also raised concerns in this regard because these drugs always have side effects. They are associated with the fact that controlling and managing blood sugar levels is necessary and has many benefits in preventing cardiovascular disease. As mentioned, people with type 2 diabetes are at higher risk for cardiovascular disease, which increases mortality in people with diabetes (Nenoi et al., 2011).
Depression is one of the most common psychiatric disorders that leads to significant complications for the active stratum of society, which can significantly reduce patient performance in all areas of occupation, social, and family relationships. Half of the patients with depression are in the age range of 25 to 65 years and epidemiological findings indicate an increase in the prevalence of depression in under 20 years of age. Depression is a disorder in the presence of a depressed mood for at least two weeks, which is usually associated with decreased concentration, difficulty in decision-making and irritability or psychomotor slowness, guilt, and thoughts related to death. Various factors have a role in the development of depression, including biological factors such as serotonin nervous systems, norepinephrine, dopamine, genetics, psychosocial factors such as various life events, and recently various internal stressors such as changes in serum cholesterol levels, triglycerides, sugar and coagulation factors are involved in depression. Depression, in addition to causing physical problems and exacerbating some symptoms such as pain, affects and reduces the ability and function of the individual. Also, the power of decision-making is stripped and the ability to take care of oneself is lowered, resulting in the independence of the individual and creates dependence, disability, and lack of self-confidence (Mirsifi Fard and others, 2018).
Changes in the interactions of neurotransmitters in the serotonergic, dopaminergic, and adrenergic nerve pathways manifest themselves in the form of depressive symptoms.
According to the above, this study was performed to investigate the effect of eNOS gene expression on diabetes-related depression in patients with type 2 diabetes referred to Shariati Hospital - Endocrinology and Metabolism Research Institute of Tehran University of Medical Sciences.
Methods: This study is of case-control type. The participants of this study are those who were referred to the diabetes clinic of Dr. Shariati Hospital. A demographic and clinical information registration questionnaire was completed for each candidate with the cooperation of the treating physician. The case group includes (50) people with type 2 diabetes and depression and the control group includes (50) people with type 2 diabetes and no depression. People with type 2 diabetes will have people with a fasting blood sugar of 6 126 mg/dl, which was confirmed during repeated tests, and people with diabetes will have no complications of diabetes such as retinopathy, neuropathy, and nephropathy. Confirmation of depression will be done with the opinion of the treating physician.
After completing the questionnaire and consent form, the participants in the study were sampled in tubes containing EDTA. After sampling, RNA extraction was performed using the TRIzol method and qualitative analysis of RNA extracted by gel electrophoresis and also quantitative analysis of RNA extracted by nanodrop device. GAPDH and eNOS primers were then designed. Then, PCR reaction and cDNA synthesis were performed to amplify the desired fragments, and finally, real-time PCR was performed to evaluate the expression of the desired gene. SPSS software was used for data analysis. A P-value less than 0.05 was considered statistically significant.
Specifications of Designed Primers
Table 1 shows the characteristics of primers designed to evaluate the effect of eNOS gene expression on diabetes-related depression in patients with type 2 diabetes (Abbaszadeh Goodarzi, 2015). The primer was designed by experts from the Endocrine and Metabolism Research Institute of Shariati Hospital.
For Real-Time PCR of 10 l volume (cDNA), 5 μl was removed (not diluted) and stored at -80 ° C after the ThermoCycler was completed. DEPC was added to the remaining 5 μl of water and a total of 20 μl was used for Real-Time PCR. The Real-Time PCR used in the Endocrinology and Metabolism Research Institute is a model (LightCycler ® 96 for Roche).
In this study, eNOS specific primer was used and GAPDH primer was used as a positive control for cDNA synthesis. Real-time PCR was used to compare the pattern and expression of the eNOS gene between the two groups (patients with type 2 diabetes with depression and without depression). In this method, after preparation and extraction of RNA and cDNA synthesis using Reverse primer and cDNA synthesis, a complementary DNA strand was made and samples obtained at a concentration of 500 ng / μl were used as a model for Real-Time PCR. The reaction was performed using SYBER Green PCR. How to combine the materials to make the reaction mixture was done according to the table below (Table 2).
Results: Quantitative Results Of RNA Extraction From Blood Using A Nanodrop Device
To evaluate the purity of the extracted RNAs, the absorption ratio at 260 and 280 nm was used by the nanodrop device and minimal protein contamination was used. In Table 4, the quantitative results of RNA extraction of several samples can be seen.
Qualitative Results Of RNA Extraction From Blood Using Electrophoresis
In addition to quantitative results, gel samples were taken to ensure the presence of RNA and the absence of DNA contamination. After viewing the images, the accuracy of the work was ensured.
Results of cDNA synthesis using GAPDH (Housekeeping Gene) primer
After ensuring the quality of the extracted RNA, a complementary DNA strand (cDNA) was made from the samples using the reverse transcriptase (RT) enzyme. The quality of cDNA was evaluated by RT-PCR technique and using GAPDH primer to ensure the presence of cDNA and DNA contamination. The results are shown in Figure 2.
Check the result of Real-Time PCR
Evaluation and comparison of eNOS gene expression in patients with Real-Time PCR technique
The present study was performed to evaluate the expression of nitric oxide synthase gene in type 2 diabetic patients with depression and non-depressive type 2 diabetic patients. The result of eNOS gene expression by Real-Time PCR showed that in patients with type 2 diabetes with depression compared to patients with type 2 diabetes without depression, the expression of this enzyme gene was increased which is not statistically significant ( p> 0/05) (Figure 3)
Statistical analysis results
The results of eNOS gene expression are shown in Figure 4-a. As the expression of the eNOS gene has increased in the case group compared to the control group, this increase is not statistically significant (p> 0.05). Also, in the histogram of Figure 4-b, it can be seen that the expression of nitric oxide synthase gene has increased in the case group compared to the control group, which is not statistically significant (p> 0.05).
Conclusion: Multiple studies show that depression is more common in patients with type 1 and type 2 diabetes than in healthy people. Worldwide, depression in diabetics is linked to the culture or society of countries. It is reported that 26% of patients with diabetes worldwide have depression. In these patients, the level of quality of life has decreased so much that the resulting mortality is increasing (Aswar et al., 2017).
Diabetes mellitus such as body mass index (BMI), family history of diabetes, smoking , physical activity, diet, and alcohol consumption remain significant. Also, in depressed diabetic patients, blood sugar control is more difficult, and diabetic complications are more. In Iran, many studies have been conducted to investigate the prevalence of depression in diabetic patients and the presented statistics are reported to be between 11-91% different. Therefore, a structured review of all documents and their combination can lead to a more complete picture of the dimensions of this problem in Iranian society (Azami et al., 2017).
According to various studies, 15-32.5% of diabetics suffer from depression. The lifespan of people with diabetes is clearly higher than that of non-diabetics, with an estimated 28% risk. In fact, diabetics are 2 to 3 times more likely to develop depression than non-diabetics.
One in three diabetics suffers from a form of depression that is associated with dysfunction and reduced quality of life. Depression is a mood disorder characterized by symptoms of low mood, decreased energy and interest, feelings of guilt, difficulty concentrating, anorexia, thoughts of death and suicide, insomnia or hypersomnia, significant weight loss, and dysfunction. Depression in some diabetics can be a psychological response to the severe pain of neuropathy. There is also evidence that plasma glucose concentrations affect the mood of diabetic patients so that depression in diabetic patients is associated with poor glycemic control and the level of hyperglycemia is directly related to the severity of depression.
Research has also shown that cerebrovascular disease and cerebral ischemia due to diabetes are often associated with depression. In mild cases of diabetes, even without obvious cerebrovascular events and recurrent attacks, the presence of CNS hypoglycemia has some degree of dysfunction. This complication initially appears as a mild cognitive disorder and over time and increases in severity, usually in the form of depression. Visual impairment due to retinopathy, recurrent hospitalizations, and sexual dysfunction are other causes of depression in diabetic patients. Mental disorders in diabetic patients have a negative effect on blood sugar control and better results can be achieved in the treatment of blood sugar by treating depression. On the other hand, it seems that the course of depression in diabetic patients is more malignant than in other patients, and this indicates the need to pay more attention to the mood of diabetic patients and treat their depression.
Nitric oxide is a reactive free radical that acts as a transmitter in several processes including neurotransmission and antimicrobial and antitumor activities. This gene encodes a nitric oxide synthase that is expressed in the liver and is induced by a combination of specific lipopolysaccharides and cytokines. The nitric oxide synthase gene with three related gene-like genes is located in the Smith-Magnesia syndrome region on chromosome 17.
Nitric oxide (NO) is a messenger molecule with various functions in the body. In macrophages, nitric oxide mediates tumor and bactericidal activities. It also has nitrosylation activity and mediates cysteine-S-nitrosylation of cytoplasmic target proteins including PTGS2 / COX2. Cysteine 247 is involved in regulating the activity of the GAIT complex and possibly in multiple targets of VIM, MSN, EZR, and ANXA5. It also increases the synthesis of proinflammatory mediators such as interleukins 6 and 8 in inflammation (Jia et al., 2014).
Lipopolysaccharides cause depressive symptoms by activating microglia and inducing pro-inflammatory factors such as nitric oxide, eicosanoids, reactive oxygen species, and some inflammatory cytokines such as TNF-α, interleukins, prostanoids, and leukotrienes. Large amounts of nitric oxide are produced in response to LPS by iNOS (induced nitric oxide synthase).
It is a protein with 596 amino acid residues and its gene (eNOS) is located on chromosome 7 of the long arm. Nitric Oxide: NO.
In 2019, Motamedi et al. Conducted a study on the effect of endurance training on angiotensin and eNOS gene expression in the heart tissue of type 2 diabetic male Wistar rats. The study population consisted of 36 male Wistar rats in Three diabetic groups with endurance training (n = 12), a diabetic control group (n = 12) and a healthy group (n = 12) were randomly placed and the expression of angiotensin and eNOS gene in these groups was evaluated using Real-Time PCR technique. Their study was a case-control study. Using statistical analysis of the data, these scientists found that the expression of angiotensin and eNOS gene in the diabetic control group compared with the healthy control group showed a significant increase. Endurance exercise reduced angiotensin gene expression. And eNOS compared with the diabetic control group. The scientists went on to argue that the expression of the angiotensin gene and the eNOS of diabetic heart tissue appear to be positively affected by endurance training (Rashid et al., 2004).
In 2015, Feng et al.Investigated the protective role of sirtuin-1 against ischemic injury - myocardial reperfusion by activating eNOS in diabetic rats. They were randomly divided into control and patient groups and their study type was case-control. Finally, the expression level of the eNOS gene was examined using the Molecular Real-STime PCR technique. They went on to argue that IRT1 protects mice against ischemia-reperfusion injury by activating eNOS in diabetic rats (Feng et al., 2015).
In 2017, Biasaki et al. Conducted research on the expression of eNOS and MRP4 gene expression in high-risk type 2 NSTEMI diabetics. The study population consists of 35 sick and healthy individuals in two groups; One: NSTEMI patients with and without type 2 diabetes (n = 15) and group 2: 20 patients with type 2 diabetes without a history of ischemic heart disease and the expression of eNOS and MRP4 protein genes were examined using real-time PCR. . Using statistical analysis of the data, the researchers found that eNOS gene expression was significantly less regulated in NSTEMI patients with type 2 diabetes than in NSTEMI patients without type 2 diabetes and controls. In contrast, MRP4 gene expression is highly regulated in NSTEMI individuals without type 2 diabetes compared with controls and in NSTEMI individuals with type 2 diabetes compared with control groups. The researchers went on to argue that decreased eNOS expression and increased MRP4 expression increased cardiovascular risk in these patients (Biasaki et al., 2017).
Conclusion
In the present paper, it was found that eNOS gene expression increased in the case group compared to the control group, which was not statistically significant (p>0.05). Regarding the increase of this enzyme in the case group, it can be said that the concentration of reactive oxygen or nitrogen species such as superoxide, nitric oxide, and peroxynitrite can increase in conditions such as inflammation that inhibit the body's natural defense and antioxidant activities. In recent years, it has been shown that TB producing IL-17 plays an important role in many autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and type 1 diabetes. IL17 is a profundal cytokine whose role in diabetes is the induction of nitric oxide synthase enzyme, followed by nitric oxide release, which causes the destruction of β cells. A 2001 study by Suzuki et al. showed that serum nitric oxide levels were higher in patients with depression than those without depression. Several studies have also shown that nitric oxide synthase enzyme plays an important role in the pathogenesis of depression by producing nitric oxide. Nitric oxide is composed of L-arginine with the help of nos enzyme and in turn activates the dissolved guanylate cyclase enzyme (sGC), which is responsible for converting guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP). The findings suggest that excessive levels of cGMP may cause a state of depression-like and that lowering its level may cause antidepressant activities. In addition, cGMP is decomposed into guanosine monophosphate (GMP) with the help of phosphodiesterase (PDE). Therefore, in speculation, it can be said that inhibiting the phosphodiester enzyme using its inhibitors may increase cGMP levels and cause depression. Therefore, it can be concluded that increasing the expression level of this enzyme is associated with depression and is not associated with diabetes. (Suzuki et al., 2001, Lee et al., 2006)
Finally, for further investigation, it is suggested that:
• Use more samples to achieve better results.
• It is recommended that this test be performed on people with non-diabetic depression to clarify the ambiguities of the test.
Keywords: Gene expression, eNOS, Type 2 diabetes, Depression, Real-Time-PCR