Type 2 diabetes is a metabolic disease, associated with a wide range of co-morbidities and complications, including retinopathy and loss of vision, nephropathy and end-stage renal disease, peripheral neuropathy, cardiovascular diseases as well as lower extremity amputations due to diabetic foot. A high prevalence rate of diabetes mellitus was noted in Saudis (23.7%) [1]. Since diabetic neuropathy (DN) is a common complication of diabetes and occurs in approximately 50% of diabetic patients [2], this complication was selected for investigation in the current study.
There is evidence to suggest that acquired immunity and inflammation as well as oxidative stress and apoptosis, may play a crucial role in the pathogenesis of late diabetic neuropathy [3].
Although high levels of sFas (marker of apoptosis) were detected in the serum of diabetic patients with neuropathy, it is not known whether this high level of serum of specific sFas can induce apoptosis in the human neuronal cell line. Furthermore, there is no study which shows any correlation between nerve conduction study and markers of apoptosis in diabetic neuropathy; such correlation may be useful in the future progress of diabetic neuropathy. Besides, no previous study investigated the inhibition of Fas-mediated apoptosis in neuronal cell line, by Fas blocker.
The objectives of the present study were: First, to evaluate and select diabetic patients with neuropathy by nerve conduction study. Second, to determine the role of Fas mediated apoptosis in the aetiopathogenesis of diabetic neuropathy, by measurement of apoptosis markers, induction of apoptosis by Fas and/or serum, to correlate the level of soluble Fas (marker of apoptosis) with the nerve conduction study. Third, to elucidate the role of apoptosis inhibition in prevention of diabetic neuropathy by the Fas blocker (ZB4).
A prospective clinical and experimental study was conducted. The clinical part was mainly in the form of nerve conduction study to evaluate and select diabetic patients with neuropathy. This was carried out in 28 healthy subjects (19 males and 9 females) aged 23–55 years, who served as controls. The median, common peroneal and sural nerves were stimulated bilaterally by means of the Nicolet Spirit system. Nerve conduction study was also carried out in 55 diabetic patients (18 males and 37 females) aged 13–57 years, using the same equipment as used in the study of control subjects. Sera of sFas and sFasL levels were measured by enzyme linked immunosorbent assay (ELISA) method [4]. Furthermore, serum and Fas blocker (ZB4) were used to induce and inhibit apoptosis, respectively [5]. The percentage of apoptosis was measured by flow cytometry. The correlation between the level of marker of apoptosis (sFas) and the nerve conduction study was worked out.
The findings of the present study showed that there was a significant increase of serum glucose and glycosylated HbA1c in diabetic patients 
. There was a significant difference between all the study groups in motor median nerve, amplitude, latency and conduction velocity 
. Also there was a significant difference in median nerve, sensory amplitude and conduction velocity in diabetic patients with and without neuropathy compared to controls 
. Concerning sural nerve, there were significant changes in sural nerve latency and conduction velocity in patients with neuropathy, as compared to control as well as to diabetic patients without neuropathy 
. Common peroneal motor conduction velocity decreased significantly in neuropathy patients as compared to the controls 
. However, there was no significant difference in the sensory latency of median nerve, amplitude or sural nerve as well as common peroneal nerve amplitude and latency between all the study groups.
The diabetic patients were classified according to the result of nerve conduction velocities and distal latencies into 28 diabetic patients with and 27 without neuropathy.
There was a significant increase in the sFas serum level in diabetics with neuropathy as compared to the controls and diabetic patients without neuropathy 
. Thus, serum sFas levels as well as conduction velocity of median nerve in diabetes could be considered as an apoptotic diagnostic marker for diabetic neuropathy.
The mean of serum sFas in diabetic patients with or without neuropathy positively correlated with the control 
. There were no significant differences between serum sFasL in different groups.
All parameters of the motor median nerve conduction study showed significant correlation with the serum sFas levels, especially the nerve conduction velocity 
.
Cells treated with the serum of diabetic patients with neuropathy showed significantly higher percentages of early and late apoptosis 
compared to the negative control. A 500
ng/ml Fas blocker (ZB4), antagonistic anti-Fas antibody caused significantly lower levels of early and late apoptosis 
compared to serum of diabetic patients with neuropathy treatment.
The findings of the present study confirm the role of nerve conduction studies in the assessment of diabetic neuropathy. There is a significant correlation between motor median nerve conduction velocity and the progress to develop diabetic neuropathy. The findings of the current prospective study revealed for the first time that there is significant correlation between sFas and the motor median nerve conduction study. The finding that serum sFas levels were significantly higher in diabetics with neuropathy compared to other groups indicates that serum sFas levels in diabetes could be considered as a good marker for the development of peripheral neuropathy. Moreover, the finding that the serum of diabetic patients with neuropathy could induce apoptosis in neuronal cells in vitro indicates that Fas mediated apoptosis may have a role in the aetiopathogenesis of diabetic neuropathy. However, animal and human studies are needed to establish the factors that regulate this process.
In the present work, we demonstrated for the first time that apoptosis induced by the serum of diabetic patients with neuropathy could be blocked or inhibited by antagonistic anti-Fas (ZB4) antibody. This finding confirms that the Fas-mediated apoptosis of the neuronal cell line is responsible for the degradation of the neurons in patients with diabetic neuropathy and provides evidence for the involvement of the receptor pathway in Fas-mediated apoptosis of these cells. Thus targeting and inhibiting Fas receptor offers an option for diabetic neuropathy therapy.
FULL TEXT
