Cell. Mol. Biol. (ISSN: 1165-158X)

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Cellular and Molecular Biology
Original Article

A comparative study of multiple biomarkers levels in complicated versus
noncomplicated type 2 diabetic patients
Hemin Mohamad Hussein1,*, Ahmed Alkhuzai2, Christer Janson3, Kawa Amin1,3,*
Department of Basic Medical Sciences/ Immunology, College of Medicine, University of Sulaimani, Sulaymaniyah, Iraq
Department of Surgery, College of Medicine, University of Sulaimani, Sulaymaniyah, Iraq.
3
Department of Medical Science, Respiratory Allergy and Sleep Research, Uppsala University Uppsala, Sweden
1
2

Article Info

Article history:
Received: November 01, 2023
Accepted: February 29, 2024
Published: April 30, 2024
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Abstract
The prevalence of diabetes mellitus is growing globally and the management of diabetes is a critical issue forpublic health. This study aimed to analyze the concentration of different biomarkers in patients with type 2 diabetes mellitus (T2DM) without complication, T2DM patients with complication (T2DM+C), and compared tohealthy controls (HC). For this aim, there were 164 participants: 59 T2DM, 60 T2DM+C, and 45 HC. Venous
blood was collected and the levels of Hemoglobin A1C (HbA1C), fasting blood glucose, Interleukin-31 (IL31), IL-35, glutamic acid decarboxylase antibody (GADA), developmental locus-1 (Del-1), fibroblast growth
factor-9 (FGF-9) and FGF-18) and lipid profile (total cholesterol, low-density lipoprotein (LDL), high-density
lipoprotein (HDL) and triglyceride) were analyzed. Results showed that IL-31 was significantly higher in
T2DM compared to HC (p<0.0001), and compared to T2DM+C (p<0.0001). IL-31 was significantly lower
in T2DM+C than HC (p=0.009). The level of serum GADA was significantly elevated in T2DM compared toHC (p=0.0009), and T2DM+C (p=0.03). There was a significant correlation between (IL-31, IL-35, GADA,
Del-1, FGF-9 and FGF-18). The duration of having diabetes was significantly longer in T2DM+C compared to
T2DM (p<0.0001). However, there was no significant difference in the level of HBA1C% between T2DM+C
and T2DM patients (p=0.98). In conclusion, there were significant differences in biomarker concentrations
between all three groups. This indicates that the monitoring of multiple biomarkers may be of value in thecontrolling of T2DM in the future.
Keywords: DEL-1; Diabetes; FGF-9; FGF-18; GADA; IL-31; IL-35; T2DM.

1. Introduction
Type 2 diabetes accounts for 90–95% of the estimated 347 million cases of diabetes diagnosed globally. The
prevalence of type 2 diabetes mellitus (T2DM) is progressively growing with the improvement of people's living
conditions, the aging of the population, and changes inpeople's lifestyles [1]. Diabetes-related persistent highblood sugar levels, known as chronic hyperglycemia, canlead to both small blood microvascular and macrovascular
problems, ultimately leading to mortality. Notwithstanding these established repercussions, a significant numberof individuals are unable to effectively control their blood
glucose levels. Approximately 50% of patients with T2DM
successfully reach one of the glycemic, blood pressure, or
lipid targets, whereas less than 20% manage to fulfill all
three simultaneously. Hyperglycemia and impaired glycemic control can be attributed to medication side effects,
pharmacological interactions, other medical conditions,and hospitalizations [2,3].
The development of T2DM is marked by a gradual decrease in the effectiveness of insulin (insulin resistance) intissues, and the reduced ability of pancreatic β cells to pro-

duce enough insulin to counteract the insulin resistance.
This results in high blood sugar levels. Previous studies
have indicated a complex interaction between oxidative
stress and inflammation. The onset of T2DM is facilitated by intricate interactions between environmental factors
and genetic variables, specifically numerous susceptibility
genes [4].
Chronic diabetic patients have persistent glutamic aciddecarboxylase antibodies (GADA), which are unaffectedby the patient's age at disease onset. Therefore, identifying
GADA is the most reliable indicator of adult-onset autoimmune diabetes. However, due to its high cost and restrictedavailability, the GADA test has yet to be widely utilized
in primary care fields [5]. There is enormous variability
in autoimmunity and β-cell dysfunction in T2DM, withGADA positivity and high C-peptide related to early insulin initiation and GADA positivity and low C-peptide increasing the risk of severe hypoglycemia. To improve theaccuracy of categorization and treatment in T2DM, several tests are required [6].
Cytokines are critical immune mediators produced in
an ordered process during the infectious and inflamma-⁎ Corresponding author.
E-mail address: hemin.hussein@univsul.edu.iq (H. M. Hussein), kawa.amin@medsci.uu.se (K. Amin).
Doi: http://dx.doi.org/10.14715/cmb/2024.70.4.8
45Cytokines in type 2 diabetes mellitus.

Cell. Mol. Biol. 2024, 70(4): 45-52

tory response, which has a particular function in cell-tocell contact. Several cytokines such as interleukins, chemokines, colony-stimulating factors, interferons, tumor
necrosis factors, and transforming growth factors are included in the cytokine superfamily, all of which exhibit
various immunological and biological functions [7].
The developmental endothelial locus-1 (DEL-1) isreleased by tissue-resident cells, including some subsets
of macrophages endothelium, and mesenchymal stromalcells. DEL-1 can interact with a variety of integrins, suchas integrins β2 (αLβ2 and αMβ2) and αv (e.g., αvβ3), as
well as phospholipids. DEL-1 may be a possible therapeutic target due to these interactions, which help it control
critical immunological processes that impact inflammatory and autoimmune illnesses in the preclinical research[8].
Fibroblast growth factors (FGF) are a group of peptide
cytokines, including 23 members with varied functions.
FGFs control several biological and cellular activities,
including skeletal and embryonic development, tumor
proliferation, inflammation, tissue formation, blood vessel formation, tissue healing, growth, and cellular specialization [9,10]. Furthermore, FGFs play significant roles
in adult tissue repair and damage response [11,12]. The
FGF family, comprising FGF-9, 16, and 20, includes the
fibroblast growth factor FGF-9 subfamily. Due to their highdegree of sequence homology, these molecules may sharesimilar metabolic functions [13]. The exosome-associatedgene FGF-9 was first found in human glioma cells, and it
has subsequently been discovered that this gene participatesin the control of glia in the central nervous system. Recent
studies have shown that FGF-9 is now understood to be
essential for the growth of malignancies [14]. The FGF-8
subfamily has two other members, FGF-17 and FGF-18,
and they have different expression patterns during embryonic development based on similarities in their aminoacid sequences [15]. FGF-18 controls chondrogenesis andosteogenesis [16,17]. In addition, FGF-18 expression was
upregulated in colon [18], ovarian [19], and breast cancer[20].
IL-31 and IL-6 are related as they belong to the samefamily, commonly grouped due to their pro-inflammatory effects and a similar signaling route that activates the
gp130 receptor subunit [21]. Different kinds of cells including helper T cell 2, macrophages, eosinophils, basophils,
dendritic cells, fibroblasts, and intestinal and pulmonary
epithelial cells are immune and nonimmune cells that can
produce IL-31 [22,23]. IL-31 modulates signaling and affects several biological processes, including the inductionof pro-inflammatory cytokines, cell proliferation, and tissue remodeling. [24]. It still needs to be determined how
IL-31 works physiologically. IL-31 has been linked toseveral inflammatory problems such as atopic dermatitis[25], inflammatory bowel disease [26], and asthma [27].
Furthermore, In mice, the cytokine Interleukin 31, generated by activated T cells, causes dermatitis and cutaneous
inflammation [28]. IL-31 was detected in both blood andvitreous specimens obtained from individuals diagnosed
with proliferative diabetic retinopathy, indicating that IL31 might have a significant role in the development of diabetes [29].
IL-35 is discovered by Collison [30] and Niedbala [31]recently. It belongs to the interleukin-12 family. IL-35 hasanti-inflammatory and immunosuppressive capacities. It

is composed of two subunits, IL-12α (also known as IL12p35) subunit, and Epstein-Barr virus induced 3 (EBI3)
subunit [30,32]. IL-35 has a significant function in the etiology and development of inflammatory and autoimmune
disorders; all of these link adaptive and innate immune
responses [33]. IL-35 is primarily created by T-regulatory
cells, B-regulatory cells, and antigen-presenting cells. IL35 can stimulate the development of Treg and Breg. On
another side, it can inhibit the activity of macrophages andeffector T lymphocytes, mainly helper T cell 1 and helper
T cell 17 [34].
Increased knowledge about the role of cytokines in
T2DM may lead to the development of improved tools
for diagnosis and prognosis as well as new targets fortreatment. The current research aimed to evaluate serum
concentrations of GADA, DEL-1, FGF-9, FGF-18, IL31and IL-35 in T2DM patients with and without complications compared with participants without diabetes.
2. Materials and Method
2.1. Study design
Cross-sectional and Comparative Study.
2.2. Study population
The study was performed in accordance with the World
Medical Association's Code of Ethics (Declaration of Helsinki) for studies involving human beings. The research
protocol (No. 75, Date 18/5/2021) was authorized by the
Ethical Committee of the Sulaimani University, Collegeof Medicine. Prior to their participation in the study, eachsubject provided written informed permission.
The study was started from May 2021 to February
2023. Patients with T2DM have participated in this study.
Diagnosis of diabetes was performed by a specialist physician (Internal physician and endocrinologist) based on
the diagnostic criteria for T2DM, and they were admitted
to the Diabetes and Endocrine Center, Shar Hospital, andprivate healthcare sectors in Sulaymaniyah City. The study
approached 277 subjects, with ages ranging from 30-80
years. Among them, 164 individuals were selected and theother 103 were excluded based on exclusion criteria andmissing data. The exclusion criteria were systemic steroid
use, other autoimmune diseases, smoking, alcoholism, andpregnancy.
The individuals were grouped as 59 T2DM patients
without complications (female 30, male 29), 60 T2DM
patients with complications (female 32, male 28), and 45
subjects representing healthy controls (HC) (female 22,
male 23). The group with complications had one of thefollowing: diabetic foot ulcer (DFU), diabetic retinopathy
or diabetic peripheral neuropathy (DPN). The HC wereindividuals without diabetes and other diseases including
hypertension.
2.3. Sampling
Venipuncture was performed to collect blood frompatients and controls, and collected in two different test
tubes; one with gel and clot activator to obtain serum andone with EDTA for whole blood. The serum tubes were left
in the rack in a vertical position without shaking for 10 to
15 minutes to permit the blood to clot, then centrifuged (6minutes, 5000 rpm) to separate serum from clotted parts.
Serum samples were divided into two parts, the first partwas used to perform several tests including fasting blood46

Cytokines in type 2 diabetes mellitus.

Cell. Mol. Biol. 2024, 70(4): 45-52

glucose (FBG), hemoglobin A1C (HbA1C), triglycerides,
total cholesterol, low-density lipoprotein (LDL) cholesterol and high-density lipoprotein (HDL) cholesterol, while
the second part was divided into three aliquots and stored
at -70oC until testes by Enzyme-Linked Immunosorbent
Assay (ELISA) for different biomarkers (GADA, DEL-1,
FGF-9, FGF-18, IL-31, and IL-35).

out the linear association between two variables and detectcorrelations among biomarkers, parameters, BMI, age,
symptom score, and duration of diabetes. A significancelevel of 0.05 or lower was employed as the threshold for
determining statistical significance.
3. Results
3.1. Patients' characteristics
The characteristics of the individuals who took part areclearly outlined in (Table 1).
Table 1 and Figure 1A demonstrate that the mean ageof T2DM+C patients was significantly higher than thatof T2DM patients and the HC group. Furthermore, theT2DM+C patients exhibited a considerably longer duration of diabetes compared to the T2DM patients, as seenby the data presented in Table 1 and Figure 1B. In addition, the HbA1C level was significantly increased in both
T2DM and T2DM+C patients compared to HC. However, there was no notable difference in the HbA1C level
between T2DM+C patients and T2DM patients (Table 1
and Figure 1C).

2.4. Analytical methods
COBAS C 111 (Roche) was used to determine HbA1C,
while COBAS C 311 (Roche) was used for FBG, triglycerides, total cholesterol, LDL cholesterol and HDL cholesterol.
2.5. Qualitative and quantitative analysis of cytokines
and FGFs
A special ELISA kit was used for each type of cytokines, fibroblast growth factors, and other biomarkers.
Anti-GAD autoantibody ELISA (Cat No. E7259Hu), Human Developmental Endothelial Locus-1 ELISA (Cat No.
E4505Hu), IL-31 ELISA (Cat No. E3254Hu), IL-35 ELISA (Cat No. E0042Hu), FGF-9 ELISA (Cat No. E5289Hu)
and FGF-18 ELISA (Cat No. E5287Hu). Serum samples
were thawed completely, and based on manufacturer instructions ELISA kits were left at room temperature for 30
minutes. The standard solution was diluted for each kit
to prepare standards from one to five. All standards were
tested alongside serum samples based on the assay procedure step by step. The optical density of each ELISA plate
was read by a microplate reader (Biotek ELX800 Microplate Reader). A standard curve was made using known
concentrations of standards with optical density, and thestandard curve was used to determine the concentration ofeach analyte in each kit by GraphPad Prism 9.0 software.

3.2. Comparing the levels of cytokines, fibroblast
growth factors, Del-1, and GADA between HC andT2DM
As shown in (Figure 2A) a higher level of serum
GADA was found in T2DM compared to HC. Likewise, asignificantly higher concentration of GADA was found in
T2DM than in T2DM+C. As shown in (Figure 2 B, C, D,
and F) there were no significant differences in the concentrations of DEL-1, FGF-9, FGF-18, and IL-35 between
the three groups. The concentration of IL-31 in blood wassignificantly elevated in T2DM patients compared to HC(Figure 2 E). The group with T2DM+C had significantly
lower concentrations of IL-31 than both those with T2DM
and the HC group (Figure 2 E). In addition, the levels of all

2.6. Statistical Analysis
The results of T2DM and T2DM+C were compared toa control group consisting of individuals of the same age
and sex who were in good health. The statistical analysis
was performed using GraphPad Prism 9.0 software. The
results were presented as the mean value plus or minus
the standard deviation (SD) and were examined using a
Mann-Whitney-U test for two groups, or a one-way analysis of variance (ANOVA). The study employed ANOVA
to compare numerical variables across the three groups.
The Spearman correlation analysis was employed to figure

Fig. 1. The difference in (A) HBA1C%, (B) Age among HC, T2DM,
and T2DM+C. (C) the difference in duration of having diabetes
between T2DM and T2DM+C.

Table 1. Demographic and clinical characteristics of diabetic patients and controls.

Parameters
M/F
Age (yr) (Mean±SD)
Duration (yr) (Mean±SD)
HbA1c Mean (%)
Family history (Yes)
Symptom scores
Hypertension (Yes)
Pills for diabetes (only)
Pills and insulin for diabetes
Insulin injection (only)
No treatment for diabetes
Pills for other diseases

T2DM (N=59)29/30
54.3± 10.8
7.9±6.1
8.8
34
4.5
18
34
11
2
12
Yes (38)

T2DM+C (N=60)28/3258.9±8.59
14.1±7.6
8.9
39
5.3
20
30
17
8
1
Yes (26)
47

Controls (N=45)23/2250.0±11.1
5.4-

p-value
0.0001<0.0001<0.0001
0.23

Cytokines in type 2 diabetes mellitus.

Cell. Mol. Biol. 2024, 70(4): 45-52

in females than males in the group of T2DM patients withcomplications (Data not shown).

BA

3.5. Correlation between cytokines, FGFs, GADA, andDEL-1 in T2DM
As shown in Table 2 in the T2DM patients, there was asignificant, positive correlation between GADA and FGF18, IL-31, and IL-35 in the group with T2DM. A significant association was also found between DEL-1 and FGF9, FGF-18, and IL-35 (Table 2). FGF-9 was statisticallysignificantly correlated with FGF-18, IL-31 and IL-35(Table 2). A statistically significant correlation was seen
between FGF-18 IL-31 and IL-35 (Table 2). Likewise,
there was a significant positive association between IL-31
and IL-35 (Table 2).

D

C

E

3.6. Correlation between cytokines, FGFs, GADA, andDEL-1 in T2DM+C
As shown in Table 3, in the group of T2DM+C patients, there was no significant correlation between GADA
and any of the other biomarkers. A significant, positive
correlation between DEL-1 and FGF-9, FGF-18, IL-31
and IL-35 (Table 3). FGF-9 was significantly correlatedwith FGF-18, IL-31, and IL-35 while FGF-18 correlated
significantly with IL-31 and IL-35 (Table 3). A strong positive association was seen between IL-31 and IL-35 in the
T2DM+C patient group (Table 3).

F

Fig. 2. The difference in the concentration of serum (A) GADA, (B)DEL-1, (C) FGF-9, (D) FGF-18, (E) IL-31, and (F) IL-35) levels
among HC, T2DM, and T2DM+C.

3.7. Correlation between cytokines and other T2DMrelated variables
There was no significant correlation between cyto-

biomarkers were compared among different diabetic patients with complications (DR, DN, and DF), and the differences were not statistically significant (data not shown).
3.3. Comparing (BMI, Cholesterol, Triglyceride, andLDL) among T2DM, T2DM+C, and HC
Regarding BMI, cholesterol, triglyceride, and LDL, nosignificant differences were seen between the three groups(T2DM, T2DM+C and HC) (Data not shown). However,significantly increased concentration of HDL was seen in
the HC individuals than in the group of T2DM patients(Figure 3).
3.4. Comparing the levels of GADA, DEL-1, FGF-9,
FGF-18, IL-31 and IL-35 in males and females
There was no statistically significant sex difference inthe concentration of GADA, Del-1, FGF-9, IL-31, and IL35 in any of the three groups (Data not shown). However,
only the serum level of FGF-18 was significantly higher

Fig. 3. The difference in the concentration of serum HDL levels
among HC, T2DM, and T2DM+C.

Table 2. Correlation between different biomarkers in patients with T2DM (Spearman correlation (rho) and p-value.

Biomarkers
GADA (ng/ml)
DEL-1
FGF-9
FGF-18IL-31

rhop-valuerhop-valuerhop-valuerhop-valuerhop-value

DEL-1 (ng/ml)
0.21
0.1211

FGF-9 (ng/L)
0.14
0.31090.54<0.0001

48

FGF-18 (ng/L)
0.52<0.0001
0.31
0.01650.37
0.0040IL-31 (ng/L)
0.35
0.00680.22
0.09910.50
0.00010.49
0.0001IL-35 (ng/ml)
0.51<0.0001
0.32
0.01300.45
0.00040.43
0.00070.38
0.0036

Cytokines in type 2 diabetes mellitus.

Cell. Mol. Biol. 2024, 70(4): 45-52

Table 3. Correlation between different biomarkers in patients with T2DM+C Spearman correlations coefficient (rho) and p-value.

Biomarkers
GADA (ng/ml)
DEL-1
FGF-9
FGF-18IL-31

rhop-valuerhop-valuerhop-valuerhop-valuerhop-value

DEL-1 (ng/ml)

FGF-9 (ng/L)

FGF-18 (ng/L)

IL-31 (ng/L)

IL-35 (ng/ml)-0.06
0.65000.07
0.57470.52<0.0001-0.09
0.48020.51<0.0001
0.43
0.00060.05
0.71820.62<0.0001
0.64<0.0001
0.44
0.00060.13
0.33810.53<0.0001
0.78<0.0001
0.39
0.00210.71<0.0001

kines and FBG, HbA1C, Cholesterol, Triglyceride, HDL,
and LDL in any of the two T2DM groups. Likewise, nosignificant correlation was found between cytokines andsymptom score or duration of diabetes. Nonetheless, inthe group with T2DM, there is a significant correlation
between IL-35 and BMI (rho=0.26, p=0.048). Similarly,
In the group of T2DM+C patients, the correlation between
age and FGF-9 was significant (rho=0.26, p=0.035,) (CI[0.01 - 0.50]).

tection and revascularization [13]. The level of FGF-18
has been studied in other diseases and was upregulated incolon [18], ovarian [19], and breast cancer [20]. FGF has
shown potential therapeutic benefits in diabetes by regulating metabolic processes [43], improving insulin resistance, and promoting adipose tissue homeostasis.
In the present investigation, a significantly higher
concentration of serum IL-31 was detected in T2DM compared to HC while the difference was not significant. Until
now, this is the first study in which the blood level of IL-31
was studied in T2DM and T2DM+C. IL-31 has been studied in other diseases, and a higher blood concentration of
IL-31 was reported in the serum of individuals with cutaneous lesions and people with atopic dermatitis comparedto HC [44,45]. However, surprisingly, we found a significantly lower level of serum IL-31 in T2DM+C comparedto T2DM and HC. In accordance with the current study,
the levels of other inflammatory cytokines were higher in
HC compared to T2DM+C, [46]. The precise mechanismof IL-31's involvement in diabetes remains incompletely
clarified, therefore more research is needed to understandits potential involvement in T2DM. Similar to other proinflammatory cytokines, we imagine that IL-31 might participate in the initiation of inflammation during diabetes.
In the current study, a higher concentration of IL-35
was found in the serum of T2DM and T2DM+C compared to HC. Studies about IL-35 in T2DM and T1DM
are limited. A recent study found that the difference in theserum concentration of IL-35 between HC and T2DM was
not significant [47]. In line with our research, patients with
T1DM exhibited elevated levels of IL-35 in their serum[48]. IL-35 might affect the progression of T1DM and
T2DM. It was hypothesized that IL-35 would reduce inflammatory cytokine production [49]. It has been shownthat IL-35 manages the suppression of Tregs, blocks Th17
cell differentiation, and thus tends to reduce the continuous
immune damage of beta-cells [50].
There was a positive correlation between GADAs andother biomarkers in T2DM. Studying these biomarkers is
new, therefore finding related work is limited. Similar tothis finding, recent research observed a positive correlation
between GADAs and (IL-1β, IL-3, and IFN-γ) in T1DM
and T2DM patients [35]. In agreement with this study, asignificant correlation was observed between GADA and(IL-6 and IL-1β) in T2DM in a previous study [36].
In T2DM and T2DM+C, positive correlations were
found between DEL-1 and other biomarkers (FGF-9,
FGF-18, and IL-35). In addition, in T2DM+C, there was

4. Discussion
The main findings of this research were that there weredifferences in biomarker levels among all three groups
studied. For many of the biomarkers, the highest levels
were found in the group with T2DM without complication
while somewhat surprisingly the group with T2DM withcomplication often had levels that were even below theHC group.
In the present study, a significantly higher level of serum GADA was found in T2DM compared to HC. This iscompatible several earlier findings [35,36]. A significantly
higher concentration of GADA was found in T2DM compared to T2DM+C. Moreover, in both T1DM and T2DM,
GADA can have pathological effects such as neurologicalsyndromes [37], and the destruction of insulin-producing
pancreatic cells in T1DM [6]. GADA positivity is an indicator of the course of diabetes, and it can also be detectedin non-diabetic patients with autoimmune diseases [38].
In the current study, a higher level of DEL-1 was foundin the serum of T2DM and T2DM+C compared to HC,
while the differences were not significant. So far, this is
the first study in which the serum DEL-1 was estimated in
T2DM, and T2DM+C compared to HC. Recent studies determined that DEL-1 participates in the host inflammatory
response at various stages from neutrophil production,
their recruitment and their eventual clearance by macrophages during the resolution of inflammation [39,40].
Del-1 is participated in the control of inflammation andangiogenesis, and it may be related to diabetes through itsrole in the regulation of endothelial cell function and its
association with T2DM.
Higher level of FGF-9 and FGF-18 was found in theserum of T2DM and T2DM+C compared to HC, while
the differences were not significant. According to previous
findings, FGF-9 may serve as a novel immuno-associatedand prognostic biomarker for ovarian cancer [41,42]. Furthermore, a recent study has concluded that FGF-9, FGF16, and FGF-20, may one day be used to treat cardiopro49

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Cell. Mol. Biol. 2024, 70(4): 45-52

a positive correlation between DEL-1 and IL-3, while thedifference between DEL-1 and IL-31 in T2DM was not
significant. However, a previous study found a negative
correlation between salivary Del-1 and IL-17 expression
levels in T2DM patients with periodontitis [51]. We expectthat DEL-1 seems to be involved in the control and resolution of inflammation. Previous studies showed that DEL-1
stimulates macrophage efferocytosis [39] and inflammation clearance, and it suppresses inflammation [52].
We found a positive correlation between FGF-9 andFGF-18 in both T2DM and T2DM+C. Further research
may be required to discover the connection between FGF9 and FGF-18. Moreover, in this study, we found that in
T2DM and T2DM+C, there was a correlation between
FGF-18 and other biomarkers (IL-31 and IL-35). In thisstudy, we found that in both groups T2DM and T2DM+C,
there was a positive correlation between FGF-9 and IL31. In contrast to our finding, a previous study claimed
that FGF-9 therapy significantly decreased the levels of
circulating pro-inflammatory cytokines, M1 macrophage
differentiation, and invading monocytes [53]. In the present study, a significant positive association was observed
between FGF-9 and IL-35. Similar to this finding, a recent
study on mice demonstrated that the release of anti-inflammatory cytokines was also boosted by FGF-9 therapy [53].
According to the situation, FGF-9 and FGF-18 may havepro-inflammatory [54,55], or anti-inflammatory effects[53,56].
Interestingly, the current study found a significant
correlation between IL-31 and IL-35 in both groups ofpatients T2DM and T2DM+C. However, there is limited
information on the correlation between IL-31 and IL-35.
We assume that during diabetes, the initiation of inflammation is promoted by several pro-inflammatory cytokines containing IL-31. This inflammation might cause
damage, and because of that anti-inflammatory cytokines
such as Il-35 will be produced to reduce undesired effects
of inflammatory reactions. Additional investigations may
be required to fully understand the relationship between
IL-31 and IL-35.
In the present study, the level of serum HDL was significantly lower in the blood of T2DM patients compared to
the HC group. A similar result was recorded by a previous
study as they found decresed levels of HDL in the blood of
T2DM patients [57,58]. Recent findings suggest a connection between HDL levels and the development and prognosis of T2DM. Hyperglycemia causes a decrease in levels of HDL and impairs it’s function. This occurs throughchanges in the proteins and lipids present in HDL particles.
Consequently, decreased levels of HDL and altered HDL
functionality have a negative effect on the functioning ofvital organs involved in maintaining glucose balance, suchas the pancreas and skeletal muscles [59].

risk for acquiring diabetes and its related microvascularand macrovascular problems.
Interest conflict
All authors declare that they have no conflict of interest.
Consent for publications
The author read and approved the final manuscript for
publication.
Ethics approval and consent to participate
The study was carried out by the Code of Ethics of theWorld Medical Association (Declaration of Helsinki) for
experiments involving human subjects. The Ethical Committee of the Sulaimani University, College of Medicine
approved the research protocol (No. 75, Date 18/5/2021),
and written informed consent was obtained from each subject before his enrolment in the study.
Availability of data and material
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Authors' contributions
All authors contributed to the design and implementationof the research, to the analysis of the results and to thewriting of the manuscript.
Funding
No funding was received for conducting this study.
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5. Conclusion
There were significant differences in biomarker
concentrations among each of the three groups. This indicates that the monitoring of multiple biomarkers may beof value in the management of T2DM in the future. Furthermore, our findings suggest that the levels of cytokines
could decline as the duration of the disease advances. It
has still to be discovered whether this diversity is causedby the illness process, the prescribed drugs, or if patientswith lower levels of inflammatory mediators are more at

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