Exploring Interleukin-6 ,CRP in cellphone addiction and depression: a case control study in the precincts of medical college

Cellphone technology has tremendously grown in its market and uses in the last decade. But it's overutilization has led to the development of new problems also. Objectionable cellphone use can be accounted for in the form of technological addiction, which can develop depression, anxiety, and other health problems. Depression is the third leading cause of global disease burden if it continues; by 2030, it will become the leading cause of disease burden. Stress or addiction can lead to the development of psychiatric disorders or vice versa. These stressors lead to the neuroinflammatory response, resulting in an exaggerated response to subsequent pro-inflammatory challenges. This study was done with an objective to explore levels of Interleukin-6 and CRP in smartphone-addicted and depressed individuals. Seventy-five cases and 75 healthy controls were selected for the study. Depression was diagnosed by Hamilton depression scale (HAMD) and cellphone addiction was evaluated by smartphone addition scale-short version (SPAS) and their serum sample was evaluated for IL-6 and CRP according to protocol. Overall Mean score of HAMD in cases was 12.21 and in controls was 4.68. Mean score of SPAS scale in cases and controls was (37.75, 17.43) respectively. Mean IL-6 levels of cases and controls (21.03±35.85, 11.07±13.61 pg/ml) respectively with the significance of (p=0.026). Higher levels of the systemic inflammatory biochemical marker IL-6 in cases are associated with depression. Smartphone addiction scores are in correlation with depression scores but not in association with interleukin-6 or CRP levels. Females of a young age are more prone to depression and smartphone addiction together. Early assessment and diagnosis could be helpful in preventing further more damage to social and mental vicinity of individuals. Inflammatory pathways may provide important new interpolation and anticipation targets for these ailments.


INTRODUCTION
The most effective mode of information and communication technology in the present era is the mobile phone/smartphones, the use of which has grown tremendously in the past years. Current generations are thriving on cellphone/smartphone technology so much so that, even a toddler lullabies by parents have been replaced by a smartphone. Smartphone utilization and bene its are such enormous that the potential threat, lying just beneath this glori ied instrument, has been masked. Objectionable cellphone use can be accounted for in the form of technological addiction, which is now addressed by medical, educational and social society as well. This phone addiction can develop anxiety, irritability, sleep disturbances, shaking, insomnia, and even illusions (Hassanzadeh and Rezaei, 2011). Research of Thomee et al. concluded that problematic and excessive use of mobile phones is in association with anxiety, sleeplessness, depression, psychological misery, and unhealthy lifestyle (Thomée et al., 2011). physical, bodily and emotional or mental problems have been reported from cell-phone abuse, including rigidity and muscle pain, fatigue, dryness, blurry vision, irritation, or ocular redness (Gutiérrez et al., 2016).
Depression is the third prominent cause of worldwide disease burden, accounting for 4.3% of total disability-adjusted life years, in terms of public health signi icance. If the trend continues, by 2030, it will become the chief reason of disease burden (Pattanayak and Sagar, 2014). Individually, it affects the mental and emotional wellbeing, compromise with the overall quality of life and may intensify the risk of other medical ailments. It also adversely affects the job and family social life, it leads to product damage and economic burden (Pattanayak and Sagar, 2014). Depressive disorders can affect any person irrespective of age, gender or community residing in urban and rural areas or slums. National mental health survey reported a lifetime prevalence of 5.2% of depression in India (Gururaj et al., 2016). Diagnostic and Statistical Manual of Mental Disorders,, includes symptoms and the diagnosis of major clinical depression (MCD), behaviour addiction (internet abuse/ gambling). Besides these, smartphone addiction has also been in the queue to be added in DSM5 (Tolentino and Schmidt, 2018). Again depression not only causes excessive mental trauma but also intrudes upon vital biological processes just like termites eating inside a log, depression regulates in lammatory pathways, metabolism pathways, autonomic function, neuroendocrine regulation, sleep and appetite (Gold et al., 2015). It has a cause and consequence relation with several noncommunicable diseases, substance abuse disorders and dietary disorders (Amudhan and Gopalkrishna, 2016). Stress or addiction in life can lead to the development of psychiatric disorders or vice versa. These stressors lead to the neurological and in lam-matory responses, resulting in an exaggerated reaction to subsequent pro-in lammatory challenges. Both acute and chronic stressors are found in association with elevated peripheral biochemical markers of in lammation (Lurie, 2018).
IL-6 is a small but multifunctional protein that can be released from many tissues and organs including blood cells, endothelium, epithelium, adipose tissue, astrocytes, microglia and neurons (Rossi et al., 2015). IL-6 is chie ly branded as a pro-in lammatory cytokine, but it also has anti-in lammatory properties (Wolf et al., 2014). Recent researches in both preclinical (Hodes et al., 2014) and clinical models (Khandaker et al., 2014) has suggested a functional role for IL-6 in the expansion of depression and potential for targeting it to treat depression in humans. N.L. Nishuty found signi icantly raised concentrations of serum IL-6 in Major depressive patients compared to control group (Nishuty et al., 2019) there are also contrasting studies available with no signi icant relationships (Chen et al., 2007;Becking et al., 2013). Hence the present study was proposed with the aim to explore levels of IL-6, CRP marker together in smartphone-addicted and depressive adults in the arena of medical college.

Study design
The present study was conducted in the Department of Biochemistry of Santosh Institute of Medical Sciences and Research, Ghaziabad with the collaboration of F.H medical college and hospital Etmadpur, Agra, and also it was ethically approved by the same authority. It was a case-control study conducted in January 2018-Dec 2019. The prime aim of this study was to evaluate serum IL-6 and CRP levels in both depression and smartphone-addicted individuals. Subgroup analyses were also performed to explore the effects of markers in the severity of depression. In addition, we also aimed to examine the gender differences in the expression of these proteins.

Participant population
In the present study, we observed data pertaining to patients diagnosed with depression and smartphone addiction together. The depression and smartphone addiction was assessed with the aid of widely used scales, Hamilton Depression Rating Scale (HAMD) and smartphone addiction scaleshort version (SAS-SV) (Hamilton, 1960). Healthy controls were included if they were free of these above disorders. All of them were apparently healthy (not suffering from acute infections/fever).
Total number of 75 cases and 75 controls data were included in the study.
The inclusion criteria were 1) adult patients ≥18 years of age irrespective of sex which were selected according to the protocol criteria of study design, among the staff/students, residents of FH medical college and any patient's attendants coming in OPD of the medical college.2) Individuals with both depression and smartphone addiction, and not receiving any antipsychotics. The exclusion criteria were:1) Pregnant females, 2) Any drug abusers or alcoholics. Informed consent of every participant was taken.3) individuals with any of the following associated complications like acute or chronic infections, antidepressant or antipsychotic treatment, allergies, autoimmune disorders, systemic diseases and or immunomodulatory treatment.

Sample collection and measurements
Blood sampling for laboratory investigations was conducted, taking all necessary precautions. Fasting peripheral venous blood samples (5mL) without anticoagulants were collected by venipuncture. The sample serum was separated, stored into aliquots, and stored at -80 • C in a deep refrigerator before laboratory assays. Serum levels of IL-6 were determined with ELISA kits (Diaclone, France) in accordance with the manufacturer's instructions. Serum CRP was measured by turbidimetry method on semiauto analyser chem7. The CRP sensitivity was 0.2mg/L.

Statistical analysis
The statistical analysis was performed using the SPSS 17.0 program (SPSS Inc., Chicago, IL, USA) in Windows 10 Ultimate. The statistical analysis of the data, including the application of tests for description and analytical parametric, with binary logistic regression tests, was performed. The independent t-test was used to compare the parametric variables between the genders. Pearson correlations were used to de ine the strength of the relationships between the examined variables. The statistical signi icance was set at p < 0.05

Comparison between cases and controls
In total, 150 individuals were randomly included, in the study after inspecting and interrogating around 170 people. Those 20 person data was disquali ied on the basis of exclusion criteria .75 cases and 75 controls were extracted for study. The quali ication of cases was to have depression and smartphone addiction together irrespective of gender. Controls were simultaneously found during the process of cases subject's identi ication, as they did not have either of depression or smartphone addiction and were also apparently healthy.
T-test shows all variables are signi icant except CRP when equal variances are assumed (Table 1). The cases comprised 33 males and 42 females with a mean age of 21.68 years. Controls had 43 males and 32 females with mean 28.61 years. Age came out as a signi icant factor among both groups (p< 0.001). It was an unexpected inding. HAMD and SPAS scores are prede ined and widely accepted scales, hence signi icant. Mean score of HAMD in cases was 12.21 and in controls was 4.68. Mean score of SPAS scale in cases and controls was (37.75, 17.43) respectively. IL-6 values are signi icant (p=0.026).Mean of cases and controls (21.03±35.85, 11.07±13.61 pg/ml).

Gender wise analysis between Cases
When independent sample test and a mean of parameters in males and females of cases were analysed, AGE factor was signi icant (p=0.004), Age of females was signi icantly less than males. IL-6 levels in males were very less (10.17±15.3), compared to females (29.57±44.35) and were significant (p= 0.019). In cases, females are found to be more depressed with the average HAMD score (12.62 ± 3.54) than males with an average score of (11.7± 3.4), but values were insigni icant. SPAS and CRP were insigni icant. Mean SPAS score in males and females (37.64±5.05, 37.83±5.13). Mean CRP in male and female is (2.65±2.90, 2.67±2.74) ( Table 2).

Gender distribution according to the severity of depression
Overall, 54 cases are of mild depression, 15 of moderate, 5 cases of severe depression and only one person of very severe depression was found in this study. Mild depression is more in males    28 (84.8%), females were 26 (61%).In moderate depression, 12 females (28.6%) and three males (9.1%) were found. A severe type of depression had 4(9.5%) females and 1 (3%) male. Only one male of very severe depression was found in the study, as depicted in (Figure 1). (Table 3) is depicting multiple comparisons by Tukey HSD. The signi icant age difference between mild and moderate depression was found (p=0.036). Signi icant difference between IL-6 values was found between mild and severe depression (p=0.004). IL-6 was also signi icantly different between moderate and severe depression groups (p=0.049). CRP levels were insigni icant. SPAS scores were signi icantly different between mild, moderate and severe depression. This is a very important inding. (Table 4) depicts t-test between cases and controls among different genders. In male's age, HAMD, SPAS category are signi icant. Mean age in controls (28±8.94 years) and cases (22.55±2.86 years), this is depicting depression and smartphone addiction is more in younger males. In females, all parameters were signi icant between cases and controls, except CRP. This is another important inding in our study. AGE and HAMD score were highly signi icant (p<0.001). Average Age in controls (29.44±11.3 years) and in cases was (21±1.5 years), here also depression is trending towards younger females. Mean of IL-6 in controls (13.3±16.06 pg/ml) were lower than cases (29.57±44.35 pg/ml) and significant in females, indicating the presence of in lammation in depression and addiction. Overall IL-6 of both genders is more in cases than controls and signi icant. (Table 5) is depicting the correlation between the parameters of case series.HAMD was found in correlation with IL-6 (p= 0.032) and SPAS (p=0.023).

Pearson correlations
This study was conducted to explore the relation of in lammatory markers (interleukin-6 and CRP ) in smartphone-addicted and depressed individuals. The study gave many astonishing results; irstly mean age was signi icantly different in cases and controls. Age was signi icant between males and females of cases also. Age of males (22.55 years) was more than females (21years). It was also found signi icant between mild depression (54 cases) cases and moderate (15 cases) depression. Also, on separate gender analysis, age was signi icant between cases and controls. Thus to our knowledge, we believe to be irst in reporting, age as a signi icant factor for smartphone addiction and depression both. Our study also indicated that younger females are tending to get addicted to smartphone and eventually in depression. Inconsistent with our indings, a study by Per Hoguland also suggested that anxiety, sleeplessness and burnout were specifically severe and prevalent in younger females. Men of middle age group have a higher occurrence of mental ill-health compared to other age groups of men, with the lowest severity and prevalence in the age span 60-69 years (Höglund et al., 2020).
Some contrasting, studies by Strodal and associates suggested that there was a negligible gender difference in depression scores and in prevalence rates of depression and both were found to increase constantly with age in both genders (Stordal et al., 2001). While discussing the severity of depression, maximum cases in our study fall in the group of mild depression with age (21.4years). Highest age of the subject was found in moderate depression (23.07) and age was lower in severe (20.8) and very severe (20.0) groups. Age factor was signi icant (p=0.058) particularly, the signi icant age difference between mild and moderate depression was found (p=0.036) In accordance with our study, Csibi S research indings showed that the (20-34 years) age group had the maximum score on the problematic smartphone usage scale, next was those aged between 3-11 years and then those aged 35-50 years. The lowest scores were for those aged 11-19 years and those over 50 years of age (Csibi et al., 2019).
These indings are establishing our facts that the younger generation is more vulnerable to depression and smartphone addiction. Possible reasons for a younger generation to get addicted could be their leisure time, lazy behaviour and no target for life or education achievements, lack of parents, teacher control or ignorance could also be the factor of their falling prey to this type of addiction and depression.
In this study, we found 33 males and 42 females in cases category clearly depicting females were more depressed. The score of female depression was more than males but was statistically insigni icant. It was documented by M Kockler that women in the overall population suffered from more depressive symptoms than men and had more hunger disturbance and joylessness (Kockler and Heun, 2002).
The phrase "gender gap" is frequently used in economic, income, social or political issues between men and women but the best-documented gender gap include a mood disorder/ depression. Women tend to develop depression twice as likely as men. They also have higher amounts of seasonal affective disorder, depressive signs in bipolar disorder, and dysthymia (chronic depression). The hormonal changes that accompany menstruation or any kind of sexual or child abuse can be explanation for women being vulnerable to depression. Some experts believe in equal development and frequency in reference to develop depression, but women are more likely to be diagnosed with this disorder because men do not share their feelings. Depression shows up in dissimilarly in men as in substance abuse or violent behaviour (Harvard Health Publish-ing, 2011).
In this study, we found biochemical associations and statistical association with cases (depressed and addicted to a smartphone). It was statistically proved by having a correlation of HAMD with SPAS, and HAMD in relation with IL-6 marker, thus proving the indirect relation of smartphone addiction and IL-6.
Interleukin-6 levels were found to be signi icant between cases and controls (p = 0.026), indicating rising levels of in lammation together with depression and addiction. IL-6 levels were signi icantly different between males and females (p=0.019*) in case category, with females levels on the higher side.
IL-6 levels were also signi icant between groups (p=0.011) of the severity of depression. Signi icant differences of IL-6 between mild and moderate depression and moderate and severe depression found, which is indicating in lammation relation with levels of depression. Signi icant difference between IL-6 values was found between mild and severe depression (p=0.004). IL-6 was found an insigni icant difference among moderate and severe depression groups (p=0.049).
Study of Yoshimura showed that 51 MDD patients, plasma IL-6 levels were elevated during the acute state of MDD compared to controls, similar to our indings (Yoshimura et al., 2009).
IL-6 was found to be correlated with a depression score of HAMD.IL-6 levels of females were significant between cases and controls but insigni icant among males. CRP levels were higher in cases than controls, and were also raised in females as compared to males but were not found signi icant in the study.
It is possible that acute nerve-racking emotional event results in ampli ied levels of in lammatory markers such as IL-6 via stimulation created by nervous, endocrine and autonomic nervous system responding together, which we have discussed earlier is more in females thus explaining the IL-6 elevation. Cytokines can cross over the blood-brain barrier, thereby in luencing many facets of mood disorder pathways and pathophysiology, including neurotransmitter mechamnism, neuroendocrine function, neural plasticity, and subsequently altering the activation of the brain and affecting emotion and behaviour. Some studies have found extraordinary raised levels of serum IL-6 in patients with depressive disorder, (Lu et al., 2019). Since IL-6 induces synthesis of CRP in the liver, stress might be contributing to increasing levels of CRP via its effects on IL-6 levels. Acute stress also has the potential to induce peripheral blood mononuclear cells to migrate from the marginal pool resulting in an increased number of circulating cells. Thus, resulting in the acute increase in the number of cells producing. IL-6 may be responsible for the increase in in lammatory markers during times of trauma and stress. A luctuation in plasma volume is another potential mechanism for stress-induced upsurges in in lammatory responses. Acute negative emotions tend to stimulate reductions in plasma volume (Brummett et al., 2010). On contrast to our study, among manic/mixed Bipolar Disorder patients, higher serum IL-6 levels were observed in males than female patients (Lu et al., 2019).
SPAS scores were not found signi icant between males and female cases in our study, but females had a higher score of addcition towards the mobile phone.SPAS scores were signi icant between mild depression and severe depression cases. It was also signi icant between moderate and severe depression. SPAS were found in correlation with the HAMD score of cases. However, no correlation was found between SPAS and any biochemical marker [IL-6, CRP]. SPAS scores were also signi icant categorically between severe and mild depression and severe and moderate depression; this is a remarkable inding as it is indicating that the severity of depression is also in a relationship with smartphone addiction.
Similar to our indings study of K Demirci, discovered that the Smartphone Addiction Scale scores of women were signi icantly advanced than those of males. Depression, anxiety and daytime dysfunction marks were higher in the high smartphone use group than in the low smartphone use group. Positive correlations were established between the Smartphone Addiction Scale scores and depression stages, anxiety levels, and some sleep quality scores (Demirci et al., 2015).
On inding a correlation between the parameters in case series, we observed IL-6 was found in correlation with HAMD scores. Separately HAMD and SPAS scores are also in correlation (p=0.023) This is a very important inding of this study. Inconsistency with our study, a study by Fan et al. indicated that IL-6 levels are positively associated with Hamilton Depression Scale-17 scores for MDD patients (Ting et al., 2020).

Limitations
While this study was strengthened by the combined analysis of biochemical, behavioural and psychological factors, but it had some limitations too. Many of the items required self-reporting hence limiting true re lection of data of health status. It was a hospital-based study and was not planned on a large scale, due to inancial restrictions. Cofounders of depression were not studied in this study, which could make another research itself. Other biochemical parameters were not included, due to lack of resources. Depression, anxiety and high rates of comorbidity are signi icantly related to interconnected and co-occurring risk factors such as genderbased roles, stressors and negative life experiences and events were not discussed separately, nor evaluated in this study.

CONCLUSIONS
The present study has led us to countless important indings. Firstly IL-6 which was a key molecule of the study was found to be correlated with cases (pvalue <0.05). Secondly, the age of males and females both is lesser in cases (22.55, 21) than controls (28, 29.44) respectively. Age factor was an important inding. Statistically, it was proven that depression cases are younger than healthy controls and this is an alarm to society, social worker, health workers, scientists who are directly or indirectly involved in constructing a culture.
In the present study, females are found to be more depressed with an average score (12.62 ± 3.54) than males with an average score of (11.7± 3.4). Signi icant difference between IL-6 values was found between mild and severe depression (p=0.004). IL-6 was also signi icantly different between moderate and severe depression groups (p=0.049). Mild depression is more in males, whereas moderate and severe type is more in females. Overall, IL-6 values are signi icantly different with (p = 0.026). IL-6 and SPAS are in association with the HAMD scale of depression independently.
Younger females are more prone towards depression and smartphone addiction in our society. We strongly recommend studying depression and smartphone addiction together, especially in teenagers and adoloscents with all more possible biochemical markers. Extensive studies with different parameters cause's covariates should be taken as new targets of research.