Background
Diabetic peripheral neuropathy (DPN), one of the most common long-term complications of type 2 diabetes mellitus (T2DM) with an estimated lifetime prevalence of more than 50%, is characterized by sensory and motor neuron damage [
1,
2]. DPN is a major risk factor for foot ulcers and even amputations, resulting in serious health consequences and a heavy economic burden. Although persistent diabetes and hyperglycemia play a central role in the development of DPN, strict glycaemic control cannot eliminate the risk of DPN, suggesting a multifactorial origin in its pathogenesis. Therefore, there is an urgent need to early identify and appropriately control novel modifiable risk factors that contribute to DPN for preventing and treating DPN at an early stage.
Bilirubin, as the end product of heme catabolism in mammals, has been generally regarded merely as a waste with potential neurotoxicity at high levels leading to neurologic dysfunctions and mental disorders [
2,
3]. Consistently, Hendrickse et al. reported that serum bilirubin negatively correlated with sural and peroneal nerve conduction velocity, and the severity of autonomic dysfunction (as reflected by the number of abnormal cardiovascular reflex tests) in a group of patients with primary biliary cirrhosis [
4]. In contrast, numerous recent studies have reported that bilirubin acts as a potent cellular anti-apoptotic, antioxidant, anti-inflammatory, and immunoregulatory agent at normal to mildly elevated levels, pointing to a cytoprotective and neuroprotective effect of bilirubin [
5‐
7]. Consistent with this notion, serum bilirubin levels have been reported to be inversely associated with the prevalence and the severity of cardiovascular autonomic neuropathy (CAN) [
5] and the presence and progression of DPN including distal symmetrical polyneuropathy (DSPN) [
2,
8], and positively associated with hands and feet electrochemical skin conductance levels for reflecting sudomotor function [
9], and corneal nerve fibers morphology for reflecting small nerve fibers (unmyelinated C-fibers) dysfunction [
10] in patients with T2DM. However, the pathological mechanisms underlying the relationship between physiological serum TBIL concentrations and DPN has not been fully understood.
Therefore, the cross-sectional study was conducted to investigate the relationship between physiological serum TBIL concentrations and DPN in a Chinese population with T2DM. Moreover, the potential associations among serum TBIL and liver enzymes, metabolic parameters, inflammatory markers, and other diabetic micro- and macrovascular complications were also evaluated.
Discussion
In the present study, we found that serum TBIL levels significantly decreased in T2DM patients with DPN, and were independently and negatively associated with VPT values. We further showed that serum TBIL level was an independent decisive factor for the presence of DPN, even after multivariate adjustment. These findings suggest that serum TBIL may be a useful biomarker of DPN, and lower physiological serum levels of TBIL might be etiologically associated with DPN.
As mentioned earlier, bilirubin, including free bilirubin, albumin–bound bilirubin, conjugated bilirubin and unconjugated bilirubin, is a potent endogenous antioxidant [
5]. Serum bilirubin levels are positively correlated with the levels of antioxidative enzyme such as superoxide dismutase, catalase, and glutathione peroxidase, and its antioxidant ability is equal to or more potent than that of α-tocopherol [
5]. Bilirubin was capable of efficiently scavenging singlet oxygen, superoxide, peroxynitrite and peroxyl radicals [
1,
5,
7]. In addition, bilirubin lowers reactive oxygen species (ROS) and protects against nerve injury by inhibiting the protein kinase C and NAD(P)H oxidase pathway [
2,
5,
6,
8]. Moreover, bilirubin might exert a neuroprotective effect by inhibiting formation of advanced glycation end-product which results in nerve fibre damage by the modification of proteins or from impaired nerve blood flow and diminished neurotrophic support [
5,
6]. It is well known that oxidative stress is a major contributor in the pathogenesis of DPN [
1,
7]. Therefor, these findings suggest that high serum bilirubin at physiological concentrations might exhibit effective antioxidant activities which might play a protective role in the development and progression of DPN. Consistent with this hypothesis, we found that serum bilirubin levels were significantly decreased in T2DM patients with DPN. Moreover, serum TBIL levels were independently and negatively associated with VPT value. VPT measurement may be an useful and reliable method for early screening DPN and reflecting the clinical severity of DPN [
1]. These results, together, indicated that there may be a potential mechanistic association between decreased serum TBIL and the development of DPN. Additionally, multivariate logistic regression analysis indicated that serum TBIL was significantly associated with the development of DPN after controlling for all potential confounders. Consistently, the prevalence of DPN was decreased by 36.8% per standard deviation increase in TBIL. Further, the cutoff point of serum TBIL concentration to predict DPN was 10.75 μmol/L. Collectively, these data demonstrate that decreased serum TBIL may be associated with the development of DPN. This hypothesis is strongly supported by some previous clinical studies showing that T2DM patients with DSPN had low levels of serum bilirubin, and serum bilirubin concentrations are inversely associated with the presence and progression of DPN including DSPN in patients with T2DM, independently of classic risk factors and other microvascular complications [
2,
8].
Numerous experimental and clinical studies demonstrated that chronic low-grade inflammation as another potential factor in DPN [
1,
7,
19]. Experimental findings to date have shown that bilirubin can disturb the expression levels of complement activity, cell adhesion molecules, and suppressing differentiation of T cell [
2,
6], and the release of tumour necrosis factor alpha (TNF-α), interleukin (IL)-2 and IL-10, and also down-regulating the expression of major histocompatibility complex class-II expression in macrophages [
8]. An inverse association of bilirubin with markers of inflammation, i.e., C-reactive protein (CRP), NLR, and so on were also reported in various diseases [
2,
8,
10,
20,
21].
Together, these results suggest that bilirubin possesses potent anti-inflammatory properties in vitro and in vivo. WBC and neutrophil counts, routinely measured classic inflammation markers, are correlated with circulating CRP concentration [
14,
22]. Higher level of NLR and lower lymphocyte counts also has recently been Regarded as a dysregulated inflammatory response and increased levels of inflammation [
14,
23]. In our study, we found that T2DM patients with DPN had significantly higher levels of neutrophil and WBC counts and NLR, and lower levels of lymphocyte count. Moreover, neutrophil and WBC counts, and NLR were positive predictors of the presence of DPN, whereas lymphocyte count was a negative predictor. Our findings further support an important role of inflammation in the development of DPN. As expected, serum TBIL levels were inversely associated with inflammation markers neutrophil and WBC counts, and NLR.
Based on these findings in our study and previous ones, we extrapolate that the association between circulating bilirubin levels and inflammatory markers may provide new insights into the potential involvement of anti-inflammatory effect of bilirubin in the pathogenesis of DPN in type 2 diabetes.
Numerous experimental and clinical studies have provided solid evidence of an important role for vasculopathy in the pathogenesis of DPN [
24,
25]. The pathological basis of diabetic vascular complications including DPN is atherosclerosis. As is well known, atherosclerosis and its consequence, tissue ischaemia and endoneural hypoxia, are characterized by a state of heightened inflammation and oxidative stress [
26], while bilirubin possesses antioxidant and anti-inflammatory activities in vitro and vivo, suggesting that decreased serum TBIL may play a protective role against micro- and macrovascular complications of diabetes including DPN. In the present study, we have revealed that T2DM patients with DPN had significantly larger proportions of diabetic microvascular (DR and DN) and macrovascular (hypertension, CHD, stroke, DFU, and PAD) Complications. Moreover, T2DM patients with DPN had significantly higher levels of blood pressure, blood glucose, surrogate markers of atherosclerosis including increased ApoB/ApoA and coagulation index, reduced ApoA, and lower ABI values. Consistently, PP, blood glucose, ApoB/ApoA, coagulation index, the prevalence of other diabetic microvascular and macrovascular complications were positive predictors of the presence of DPN, especially ApoB/ApoA and the prevalence of DFU were independent decisive factors for the presence of DPN. These findings were consistent with previous studies [
2,
11,
27,
28], which imply a potential important role of metabolic and vascular disorder associated with atherosclerosis in the development of DPN. In addition, we demonstrated that serum TBIL levels were positively associated with HDL-C, ApoA and eGFR, and negatively with SBP, PP, TG, ApoB/ApoA, urinary ACR, and the prevalence of hypertension, DFU, DN and DR, and HDL-C and urinary ACR were independent factors determining serum TBIL levels, almost consistent with previous studies [
5‐
7,
10]. Surprisingly, we also found that serum TBIL levels were negatively associated with fibrinogen, and PT, INR, and fibrinogen were independent factors determining serum TBIL levels, demonstrating that serum TBIL may negatively regulate coagulation index, and might have anti-thrombotic property. Two previous studies reported a correlation between serum bilirubin and PT and APTT in 1-month-old infants and HIV-infected adults on stable antiretroviral therapy [
29,
30]. Recently, Cho et al. reported that high bilirubin concentrations were independently associated with low levels of fibrinogen and plasminogen activator inhibitor-1, respectively, in Korean subjects [
31]. More recently, Gligorijević et al. demonstrated that bilirubin and fibrinogen interact at physiological concentrations, bilirubin may exert antioxidant effect on fibrinogen, preventing its carbonylation and aggregation [
32]. Our results, together with the previously published studies, support the possibility that bilirubin may confer vascular protective and neuroprotective effects possibly due to its antioxidant, anti-inflammatory, anti-atherosclerosis, and anti-thrombotic activities, and therefore the vascular protective and neuroprotective effects of bilirubin might contribute, in part, to the relationship between bilirubin and DPN in diabetic patients.
Our study has some limitations that need to be discussed. Firstly, we could not determine the causal relationships and provide a proven mechanism for the observed association between bilirubin and DPN due to the cross-sectional nature of our study. Secondly, our study population included only Chinese Han hospitalized patients with T2DM, and therefore our findings may not be generalizable to diabetic outpatients, other types of diabetes mellitus with different ethnicity, and non-diabetic population. Thirdly, all biochemical parameters including bilirubin, in most cases, were measured only once, which may underestimate the true association because of this variability over time. However, the use of standardized methods set in a single center, and measurements taken from subjects in a fasting state should improve reliability because there will be less fluctuation in TBIL levels than in the postprandial state. Fourthly, we chosen only WBC count and differential peripheral WBC as markers of inflammation because classic markers of inflammation such as CRP, TNF-ɑ, and IL-6 are not available. Lastly, although the well known risk factors were included in multivariable analysis to evaluate the association of serum TBIL with DPN, some residual or undetected confounding effects associated with serum TBIL cannot be ruled out. Despite these limitations, the strengths of the current study include its relatively large sample size and the very well-characterized nature of the patient cohort. Moreover, this study include the strict inclusion/exclusion criteria, and thorough multivariate adjustment.
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