Association of serum biomarkers level transforming growth factor- β and tumor necrosis factor- α with diabetic neuropathy

Abstract

BACKGROUND:

Many studies have examined the role of inflammation in the development of diabetic neuropathy (DPN).

OBJECTIVE:

Evaluate the relation of the serum level of Transforming Growth Factor- $\beta$ and Tumor Necrosis Factor- $\alpha$ and development of diabetic peripheral neuropathy DPN.

METHODS:

In a case-control study, randomly selected 140 diabetic patients were included, the randomly selected patients were divided equally and matched into a case group who have diabetic peripheral neuropathy and diabetic neuropathy-free patients as a control group. For both groups whole blood sample was examined to compare for (TGF- $\beta$ ), and (TNF- $\alpha$ ) levels determination by ELISA technique.

RESULTS:

The age of the study samples ranged from 25 to 80 years with a male ratio of 1.45:1 although the sex differences between both groups were not significant. The mean levels of (TNF- $\alpha$ ) and (TGF- $\beta$ ) was significantly higher among cases group than that of controls group (254.86 $\pm$ 75.9 vs158.01 $\pm$ 50.600) for TNF- $\alpha$ and for TGF- $\beta$ (312.85 $\pm$ 62.27 vs. 217.82 $\pm$ 52.95) respectively. Both TNF- $\alpha$ and TGF- $\beta$ have high sensitivity and specificity in detection of DPN. The sensitivity of TNF- $\alpha$ was 95.7% and specificity of 61.4% area under the ROC curve (AUC) of 0.870 $\pm$ 0.029, while the sensitivity of TGF- $\beta$ was 91.4%, and specificity of 67.1 with good area under the ROC curve (AUC) of 0.891 $\pm$ 0.026 ( $P=$ 0.000).

CONCLUSIONS:

TNF- $\alpha$ and TGF - $\beta$ are significantly elevated levels in patients with DPN, these cytokines could be used as indicators for the development of DPN.

Keywords

Cytokines Diabetic Neuropathy TGF-β TNF-α

1. Introduction

Diabetic neuropathy stands as the most prominent enduring consequence of diabetic mellitus (DM). Diabetic neuropathy typically manifests as a distal sensorimotor neuropathy, characterized by diminished or lacking protective feeling in the lower extremities, resulting in a heightened susceptibility to foot ulcers, with peripheral neuropathic pain being a prevalent symptom [1].

DPN is linked to a high mortality rate and unfavorable prognosis. The exact cause of its development is not completely understood. DPN is defined by abnormalities such breakdown of nerve fibers, localized loss of myelin, or excessive growth of myelin in nerve fibers. Diabetic Peripheral Neuropathy (DPN) is thought to be linked to insufficient insulin or insulin resistance, elevated blood sugar levels, and aberrant lipid levels, resulting in increased inflammatory markers or activation of inflammatory signaling pathways [2].

Many Studies have showed that the inflammatory cytokines contribute to the development of DNP, and type 2 diabetes mellitus patients with severe nerve damage exhibit elevated levels of these cytokines [3].

Moreover, high levels of many biomarkers have been observed in Type 2 Diabetes Mellitus. Individuals with diabetes type 2 have been revealed to have higher amounts of tumor necrosis factor-alpha (TNF a) and interleukin six (the IL-6), two essential cytokine mediators, within their bloodstream in contrast to healthy individuals [4].

Also studied in people with T2DM were additional inflammatory biomarkers, such as transforming growth factor-beta (TGF-b). The markers were discovered to rise at the start of type 2 diabetes and stay high as the disease advances. The role of inflammatory parameters in the progression and consequences of type 2 diabetes (T2DM) is revealed by these findings [3].

Tumor necrosis factor-alpha (TNF- $\alpha$ ) is a one of the inflammatory cytokine that has many essential effects on different body organs and human tissue Among of its roles in the development of inflammatory and autoimmune disorders is its role as a key regulator of inflammatory reactions [5].

It significantly contributes to the development of diabetic neuropathy, in contrast to retinopathy or nephropathy where IL-6 and CRP play a more prominent role. TNF- $\alpha$ plays a confirmed function in both peripheral and central sensitization pathways, leading to the development of neuropathic pain. Long-term high blood sugar causes elevated production of tumor necrosis factor-alpha (TNF- $\alpha$ ), resulting in the formation of small and large blood vessel diseases, nerve fiber injury, and local loss of myelin [6].

The exact mechanisms by which TGF- $\beta$ affects diabetic neuropathy are not well understood, however several pathways exist. TGF- $\beta$ may induce the synthesis of extracellular matrix proteins such as collagen and fibronectin, resulting in their buildup in neural tissue and subsequent impairment of nerve function [7]. TGF- $\beta$ activate the Smad signaling pathway, which can affect the expression of genes associated with inflammation, oxidative stress, apoptosis, and autophagy. This has the potential to cause injury to nerve cells [7, 8]. TGF- $\beta$ 1 has been identified as an indicator of the ability to regenerate and polyneuropathy [9].

The study aims to compare the levels of tumor necrosis factor-alpha (TNF- $\alpha$ ) and transforming growth factor- $\beta$ (TGF- $\beta$ ) as immunological parameters among study groups of patients with and without neuropathy, as well as assess the predictive role of tumor necrosis factor-alpha (TNF- $\alpha$ ) and transforming growth factor- $\beta$ (TGF- $\beta$ ) as predictive markers for diagnosing diabetic patients with neuropathy.

2. Materials and methods

2.1 Study design and study groups

This study is a case-control. respondents met the inclusion and exclusion criteria. The sample was systematically randomly selected from attendant patients to Al-Numan Teaching Hospital including 140 type 2 diabetic patients, from October 2023 to January 2024, the patients were divided into two groups in this study, those with diabetic neuropathy and diabetic patients without neuropathy. matched groups of female and male individuals with diabetic neuropathy (43 females, 27 males) and a control group of individuals with diabetes but without neuropathy (40 females, 30 males) is included in this study. The inclusion criteria of the case group are a case of T2 DM for more than 6 months with Clinical approved diabetic neuropathy while the Inclusion criteria of the control group is a Known case of T2 DM for more than 6 months with no neuropathy. The Exclusion criteria for type 1 Diabetic patients, or patients who have specific complications diabetic ketoacidosis. Also Patients who known to have other neurological diseases.

Table 1
Baseline characteristics of the study’s sample ( $n=$ 140)

Characteristics	Study groups
	Cases ( $n=$ 70)	Control ( $n=$ 70)	Total ( $n=$ 140)	Significance
Age (years)
Mean $\pm$ SD	60.86 $\pm$ 9.640	56.53 $\pm$ 10.955	58.69 $\pm$ 10.509	$t=-$ 2.482, df: 138,
Range (min-max)	39 (41–80)	48 (25–73)	55 (25–80)	$P=$ 0.014^a
Age (In groups)
$\leqslant$ 35		2 (2.9)	2 (1.4)	$x^{2}$ : 11.291, df: 4,
36–46	3 (4.3)	12 (17.1)	15 (10.7)	$P=$ 0.023^b
47–57	27 (38.6)	21 (30)	48 (34.3)
58–68	21 (30)	25 (35.7)	46 (32.9)
$>$ 68	19 (27.1)	10 (14.3)	29 (20.7)
Sex
Female	43 (61.4)	40 (57.1)	83 (59.3)	$x^{2}:$ 0.266, df: 1,
Male	27 (38.6)	30 (42.9)	57 (40.7)	$P=$ 0.606^b

^a: Unpaired T-Test, ^b: Pearson Chi-Square Test.

2.2 Ethics consideration

The research received approval from the Medical Ethics Committee of the College of Medicine at Al Iraqi University. The official permission certificate is no (350) at 18/10/2023 , the certificate of ethical committee approval no. (F.M.S .A 105) at 31-3-2024 . Having obtained a comprehensive comprehension of the research, every participant willingly signed the informed consent form.

2.3 Data from the lab

Blood sampling was performed by venipuncture of the cubital vein, once five milliliters per sampling. Samples were taken during hospitalization, The collected material was promptly converted into a gel-filled tube and allowed to coagulate for 15 minutes at room temperature (20–25^∘C). Next, the sample was subjected to centrifugation at a speed ranging from two thousand to three thousand spins per minute for a duration of ten minutes in order to separate it and obtain the serum. the serum was put into five Eppendorf tubes and kept in a deep freezer ( $-$ 80)^∘C until ELISA analysis for assessment of Transforming growth factor – $\beta$ (TGF- $\beta$ ) and Tumor necrosis factor-alpha (TNF- $\alpha$ ). The measurements were conducted in the biochemical laboratory serum was prepared and essential material for (TGF- $\beta$ ), and (TNF- $\alpha$ ) determination by ELISA technique. TNF- $\alpha$ and TGF- $\beta$ were determined by ELISA test kits of the manufacturer Cloud-clone crop (USA).

As a measure of glycemic control the level glycated hemoglobin (HbA1C) was assessed. The glycated hemoglobin level was measured to all patients in this study by standard daily procedures in the hospital, using NycoCard Reader system which is an in vitro diagnostic test system designed to quantitatively determine the percentage of hemoglobin A1c in human whole blood.

Clinical Evaluation of Diabetic Peripheral Neuropathy (DPN). The neurological examination was performed by expert physician. The evaluation consisted of a thorough medical history and assessment of either temperature or pinprick sensation to evaluate small-fiber function, as well as the use of a 128-Hz tuning fork to assess vibration sensation and evaluate large-fiber function ,The assessment include examination of every abnormal findings seen in the limbs appearance, such as deformities, dry skin, calluses, infections, or fissures. It also assessed foot ulceration, the presence of neurological reflex these finding supported by neural electrical studies.

2.4 Statistical analysis in methods

Unpaired $t$ -test, and One Way ANOVA test were used to identify the significant differences between study groups of cases and controls regarding different quantitative parameters like age differences among study groups as well as the mean differences of immunological parameters of (TNF- $\alpha$ ) and (TGF- $\beta$ ) among case groups (unpaired $t$ -test) in addition to the mean differences of immunological parameters of (TNF- $\alpha$ ) and (TGF- $\beta$ ) among case groups stratified by disease duration (One Way ANOVA), whereas Chi-square test was used to identify the significant differences between study groups of cases and controls regarding different qualitative and categorial parameters like sex differences among study groups respectively. The study employed a logistic regression model and Roc curve to determine the most suitable threshold value of immunological indicators as predictive noninvasive markers for assessing the likelihood of neuropathy problems in diabetes patients. A significance level of less than 0.05 was employed to establish statistical significance.

Table 2
Mean comparison of immunological parameters of (TNF- $\alpha$ ) and (TGF- $\beta$ ) among case groups’ disease duration ( $n=$ 70)

Disease duration ( $n=$ 70)	Immunological Parameters (Mean $\pm$ SD)		Significance^a
	(TNF- $\alpha$ )	(TGF- $\beta$ )
$<$ 8 Years ( $n=$ 32)	271.55 $\pm$ 88.87	316.28 $\pm$ 74.26	F of TNF $\alpha$ $=$ 1.120, df: (4,1), $P=$ 0.355
8–15 Years ( $n=$ 17)	228.69 $\pm$ 63.51	311.16 $\pm$ 49.30
16–23 Years ( $n=$ 16)	253.56 $\pm$ 63.79	309.70 $\pm$ 55.39
24–31 Years ( $n=$ 4)	226.77 $\pm$ 25.06	286.78 $\pm$ 31.34	F of TGF- $\beta=$ 0.549, df: (4,1), $P=$ 0.700
$>$ 31 Years ( $n=$ 1)	298.73900 $\pm$ 0.00	386.67 $\pm$ 0.00

a: One-Way-ANOVA Test.

Figure 1.

ROC Curve of neuropathy development risk predicted by the immunological parameter of tumor necrosis factor-alpha (TNF- $\alpha$ ) among diabetic cases sample ( $n=$ 70).

3. Results and discussion

A total of 140 of 1:1 ratio of collected cases and control samples were investigated respectively following inclusion and exclusion criteria. The age of study samples was normally distributed and ranged from 25 to 80 years with a mean of 58.69 $\pm$ 10.509 with most of the sample in the age group of 47–57 years old (34.3%). The mean age of the cases group was 60.86 $\pm$ 9.640 years old mostly in the age group of 47–57 years old (38.6%), and that of controls was 56.53 $\pm$ 10.955 years old mostly in the age group of 58-68 years old (35.7%) with significant mean differences among them ( $t=-$ 2.482, df: 138, $P=$ 0.014). However, the entire study’s sample was female predominant (59.3%) with female to male ratio of 1.45:1, as well as females were dominant among cases (61.4% vs. 38.6%) and that of the control group (57.1 vs. 42.9%) respectively, but such differences were not statistically significant ( $P>$ 0.05) which reflecting the matching purpose of samples collection (Table 1).

The mean level of glycated haemoglobin (HbA1C) was significantly higher among cases group than that of controls group (8.2659 $\pm$ 1.91850 vs. 7.1907 $\pm$ 1.61363) respectively with significant differences of 1.07514 ( $t=-$ 3.588, df:138, $P=$ 0.000).

The average levels of the tumor necrosis factor-alpha (TNF $\alpha$ ) were significantly greater in the cases group compared to the control group. (254.86454 $\pm$ 75.946396 vs. 158.01620 $\pm$ 50.600267) respectively ( $t=-$ 8.879, df: 138, $P=$ 0.000) with significant differences of 96.848343. Moreover, the mean levels of transforming growth factor- $\beta$ (TGF- $\beta$ ) were higher among a group of diabetic neuropathy than that of the diabetic neuropathy-free group (312.85711 $\pm$ 62.276097 vs. 217.82637 $\pm$ 52.952765) ( $t=-$ 9.726, df: 138, $P=$ 0.000) with significant differences of 95.030743. The optimal cutoff value of tumor necrosis factor-alpha (TNF- $\alpha$ ) to detect diabetic patients with a high risk of neuropathy development was 166.67450 with a sensitivity of 95.7% and specificity of 61.4% and correctly predicted by the regression model of 78.6% with good area under the ROC curve (AUC) of 0.870 $\pm$ 0.029 ( $P=$ 0.000), (Fig. 1).

Figure 2.

ROC Curve of neuropathy development risk predicted by the immunological parameter of transforming growth factor- $\beta$ (TGF- $\beta$ ) among diabetic cases sample ( $n=$ 70).

Similarly, the optimal cutoff value of transforming growth factor- $\beta$ (TGF- $\beta$ ) for detecting diabetic patients with a high risk of complication development of neuropathy was 248.42700 with a sensitivity of 91.4%, and specificity of 67.1% and correctly predicted by the regression model of 80% with good area under the ROC curve (AUC) of 0.891 $\pm$ 0.026 ( $P=$ 0.000), (Fig. 2).

There were no significant differences seen in the mean levels of the examined immunological parameters between the cases group and disease duration. among diabetic patients with neuropathy and its several duration periods of either less than 8, 8–15, 16–23, 24–31, or more than 31 years respectively ( $P>$ 0.05).

Individuals with type 2 diabetes may eventually develop diabetic peripheral neuropathy (DPN) as time progresses. It is considered one of the common complications of diabetes more than 50% of type 2 diabetic people have Diabetic Peripheral Neuropathy (DPN) [9].

Regarding demographic distribution this study revealed that age distribution and the level HbA1c in the case group shows significantly higher than those in control group . This result was in agreement with most of the studies. The studies revealed that the prevalence of DPN increased with age and it has been observed that there is a statistically significant disparity in the mean age and the level of HbA1c of participants between the studied case group (DPN patients) and the control group (NDPN) [6, 3].

However, the study’s sample consisted mostly of females, with a female to male ratio of 1.45:1. Females were also more prevalent in both the cases and controls groups. However, these differences were not found to be statistically significant, which is in line with the findings of Mussa et al. [3].

Regarding TNF- $\alpha$ our study shows that the mean levels of tumor necrosis factor-alpha (TNF- $\alpha$ ) were significantly higher among the cases group than that of the controls group. Many studies show the same results one of the meta-analysis studies including fourteen studies and 2650 participants, the TNF -was significantly higher in DPN cases than non DPN groups [11].

Likewise, this result agreed with a recent meta-analysis study done by Sen et al. [12]. A comprehensive review and meta-analysis were conducted to evaluate the correlation between TNF- $\alpha$ and the risk of diabetic peripheral neuropathy (DPN) patients . A total of twenty-three research projects were identified that examined blood TNP $\alpha$ levels in diabetic patients with peripheral neuropathy, as compared to those without neuropathy. Out of the research conducted, 19 of them demonstrated an elevation in TNF $\alpha$ levels, while the remaining studies failed to identify any significant alteration in serum TNF $\alpha$ levels among patients with DP [12]. The optimal cutoff value of tumor necrosis factor-alpha (TNF- $\alpha$ ) for detecting diabetic patients with a high risk of neuropathy development was 166.674 with high sensitivity, and specificity and correctly predicted by the regression model of 78.6% with a good area under the curve. This result agreed with Zheng et al. [13] cross-sectional study in China about the relation between inflammatory cytokine levels among DPN and non-DPN patients. The study’s sample divided according to have neuropathy or neuropathy free as case and control groups. The DPN group consisted of individuals who were diagnosed with DPN. This group was further divided into three subgroups based on the degree of their diabetic neuropathy: mild, moderate, and severe. The researchers found that TNF- $\alpha$ , a cytokine, had a higher diagnostic value for diabetic peripheral neuropathy (DPN) compared to other cytokines. The results showed that patients in the DPN group had higher levels of TNF- $\alpha$ compared to those in the Non -DPN group. Based on the area under the ROC curve (AUCROC), optimal cut-off points, sensitivity, and specificity, TNF- $\alpha$ demonstrated a diagnostic value of over 70.0% (AUCROC $=$ 70.8%) for DPN. The sensitivity and specificity were determined to be 62.2% and 72.7%, respectively [13].

Regarding TGF- $\beta$ our study shows that the mean levels of transforming growth factor- $\beta$ (TGF- $\beta$ ) were higher among a group of diabetic neuropathy than the diabetic neuropathy-free group. This result was similar to the finding of Hussain et al. [14], who Recognized Elevated concentrations of TGF- $\beta$ 1 in the blood of individuals with type 2 diabetes mellitus (T2DM) who have neuropathy suggest a potential role in the progression of neuropathy. This finding makes it a potential biomarker for diabetic peripheral neuropathy development [14].

On the other hand, this finding disagreed with (Mussa et al., [3] study Another cross-sectional clinical investigation was undertaken in Emirate to investigate the levels of inflammatory biomarkers in patients with diabetic neuropathy who have type 2 diabetes mellitus (T2DM) [3]. The minimum age for inclusion in the study was 18 years. However, other criteria, such as having type 2 diabetes mellitus (T2DM) for more than five years, were also considered. The researchers discovered a significant decrease in TGF $\beta$ levels in patients with diabetic nephropathy (DNP) compared to patients without DNP, even after adjusting for gender, age, body mass index (BMI), and glycated hemoglobin (HbA1c). However, the study concluded that TGF- $\beta$ is regarded a predictive inflammatory sign for DPN. This difference from our study may be explained by the selection of patient who have long DM duration more than five years which is not employed in the current study [3].

The optimal cutoff value of transforming growth factor- $\beta$ (TGF- $\beta$ ) to detect diabetic patients with high risk of complication development of neuropathy was 248.42700 with high sensitivity and specificity, predicted by the regression model of 80% with good area under the ROC curve. This result is in line with a study conducted by Ybarra et al. [15]. The study focused on investigating Transforming Growth Factor Beta 1 as a biomarker for early prediction of diabetic peripheral neuropathy. In this cross-sectional study, the researchers examined the relationship between TGFbeta1 levels, for many complications type 2 diabetes mellitus (T2DM) including peripheral neuropathy. They found that TGFbeta1 is an important biomarker for screening DPN in this group. The study showed that TGFbeta1 levels were significantly higher in patients with DPN compared to those without. The integration of ROC curve evaluation using these parameters demonstrated significant sensitivity, specificity, positive predictive value, and negative predictive value in connection to the presence of DPN [15].

Regarding the association between immunological parameters and disease duration among case groups, no significant differences were identified between the means of tumor necrosis factor-alpha (TNF- $\alpha$ ), and transforming growth factor- $\beta$ (TGF- $\beta$ ) among diabetic patients with neuropathy ( $P>$ 0.05).

This finding is in line with a recent study (Sher et al., [9]). This study includes was designed to compare values of the cytokines and inflammatory markers between patient groups subdivided into three groups according to the duration of DM and: a control group and to assess the correlations between these markers with a duration of DM, it found that TNF alpha and TGF beta values did not differ statistically significantly between the groups with different durations of diabetes [9].

Ultimately, this research demonstrated that individuals with DPN have notably increased levels of inflammatory markers, such as TNF-alpha and TGF-beta. This inflammatory cytokine has the potential to serve as a valuable marker for predicting the occurrence of diabetic peripheral neuropathy (DPN).

Ethical approval

Required approval was achieved by the institute according to the Helsinki Declaration. The official permission certificate is no.(350) at 18/10/2023 , the certificate of ethical committee approval no. (F.M.S .A 105) at 31-3-2024.

Datasets/data availability statement

The data substantiating the results of this study are available and can be retrieved from the authors on demand.

Footnotes

Acknowledgments

Extended deepest gratitude to all the participants who generously contributed their time and insights to this study. Special thanks are extended to the administrative head of Al-Nu’man Teaching Hospital and all physicians for their valuable contribution and support.

Conflict of interest

No conflict of interest was declared by the authors.

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Association of serum biomarkers level transforming growth factor- β and tumor necrosis factor- α with diabetic neuropathy

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Study design and study groups

Table 1 Baseline characteristics of the study’s sample ( n = 140)

2.3 Data from the lab

2.4 Statistical analysis in methods

Table 2 Mean comparison of immunological parameters of (TNF- α ) and (TGF- β ) among case groups’ disease duration ( n = 70)

Ethical approval

Datasets/data availability statement

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
Baseline characteristics of the study’s sample ( $n=$ 140)

Table 2
Mean comparison of immunological parameters of (TNF- $\alpha$ ) and (TGF- $\beta$ ) among case groups’ disease duration ( $n=$ 70)