Sildenafil Enhances the Therapeutic Effect of Islet Transplantation for Diabetic Peripheral Neuropathy via mTOR/S6K1 Pathway

Purpose This study aimed to investigate the potential mechanism underlying the therapeutic effect of sildenafil in combination with islet transplantation for diabetic peripheral neuropathy. Methods A streptozotocin-induced diabetic mouse model was established to evaluate the effects of islet transplantation and sildenafil intervention. The mice were subjected to different interventions for 6 weeks, and histopathological staining and immunohistochemistry techniques were employed to examine the pathological changes and protein expressions of BDNF, MBP, and cleaved caspase-3 in the sciatic nerve tissue. Moreover, RSC96 cells were cocultured with islet cells and sildenafil under high glucose conditions to investigate the potential involvement of the mTOR/S6K1 pathway, BDNF, and MBP proteins. Western blotting was used to detect protein expression in each group. Results The results showed that islet transplantation can restore sciatic nerve injury in diabetic mice, and sildenafil can enhance the therapeutic effect of islet transplantation. In addition, the combination of sildenafil and islet cells significantly upregulated the expression levels of mTOR/S6K1, BDNF, and MBP in RSC96 cells under high glucose conditions. Conclusions Islet transplantation can reverse sciatic nerve injury in diabetic mice, and islet cells exhibit a protective effect on RSC96 cells under high glucose conditions via the activation of the mTOR/S6K1 pathway. Sildenafil enhances the therapeutic effect of islet transplantation, which may represent a potential treatment strategy for diabetic peripheral neuropathy.


Introduction
Diabetic peripheral neuropathy (DPN) is one of the most common complications among individuals diagnosed with type I or type II diabetes [1].DPN is characterized by distal symmetrical polyneuropathy and autonomic neuropathy.Approximately 50% of diabetic patients develop neuropathy, often without awareness of its onset due to its gradual progression.Te prognosis for DPN patients is often poor and includes a heightened risk of amputation, posing physical and psychological burdens on patients [2,3].One of the key components of myelinated nerve fbers is the myelin sheath structure composed of Schwann cells, which plays an important role in maintaining the normal structure and morphology of nerve fbers, nutritive nerve [4], and nerve injury repair and regeneration [5].Schwann cell injury is a primary pathological characteristic of DPN [6].In diabetic rat sciatic nerve tissue, nerve axons were irregular and atrophied, while the myelin sheath was uneven in thickness and damaged [7].Some studies have found that under the condition of high glucose, the apoptosis of rat Schwann cells increased and the expression of brain-derived neurotrophic factor (BDNF) decreased [8].BDNF is implicated in the expression of myelin basic protein (MBP) [9], which is essential for maintaining the normal physiological function and structure of the myelin sheath.Terefore, it is worth investigating approaches for reducing and repairing Schwann cell damage as a potential avenue for treating DPN.
Islet transplantation was reported to be an efective β-cell replacement therapy because of its good efect in stabilizing blood glucose [10].With the optimization and development of islet isolation, islet transplantation has increasingly become a practical and feasible treatment for patients with type I diabetes.A study of 84 patients who underwent islet transplantation revealed that 23 (68%) received single islet transplants, while 47 (94%) received two transplants and maintained islet activity for 12 months.In 64% of patients who received one or two times of transplant, islet function remained normal for around 6 years [11].Multiple studies have corroborated the efectiveness of islet transplantation in mitigating not only diabetes itself but also diabetic complications, including diabetic nephropathy [12], diabetic cardiomyopathy [13], and diabetic testicular injury [14].However, we note that there are few studies that delve into the specifc function and mechanism of islet transplantation in diabetic peripheral nerves.
Sildenafl, a classic phosphodiesterase-5 inhibitor (PDE5 inhibitor), is commonly used as a clinical drug for the treatment of male erectile dysfunction [15].Recent studies have also explored the potential benefts of sildenafl for the treatment of pancreatitis due to its anti-infammatory, antioxidative, and antiapoptotic properties [16].Clinical cases have reported that some patients with both erectile dysfunction and diabetic peripheral neuropathy (DPN) experienced relief of DPN symptoms after taking sildenafl [17].Experimental studies have further demonstrated that PDE5 inhibitor treatment may enhance vascular function and axon remodeling, ultimately improving the neurological function of diabetic mice with peripheral neuropathy [18].Furthermore, previous studies have demonstrated that sildenafl can provide protection to rat adrenal pheochromocytoma cell PC12 under high glucose conditions [19].In addition, sildenafl has also been found to activate mitochondrial ATP-sensitive potassium channels, which could potentially confer resistance to tissue ischemia injury [20].Moreover, a recent study suggested a possible immunosuppressive efect of sildenafl in vitro.Terefore, we aim to investigate the potential efects of sildenafl on DPN and explore the possibility of combining sildenafl with islet transplantation as a novel therapeutic approach for DPN.
Mammalian target of rapamycin (mTOR) is a Ser/Tr kinase, which participates in a number of physiological and pathological processes, such as growth, signal integration, cancer, diabetes, and aging [21].mTOR is a double-edged sword for diabetes.In diferent cells, it can promote and restrict diabetes.Studies have found that inhibition of mTOR can protect renal function and reduce apoptosis in diabetic mice [22], while activation of mTOR can restore cardiac injury in diabetic mice [23].Ribosomal protein S6 kinase 1 (S6K1) is the main downstream target of mTOR and is a member of the AGC kinase family that has been extensively studied in various metabolic, infammatory, agingrelated, and nervous system diseases and cancers [24].During the diferentiation of Schwann cells from amniotic fuid stem cells, S6K1 regulates nerve growth factor receptor (NGFR), glial fbrillary acidic protein (GFAP), central nervous system specifc protein S100 β, and other related proteins [25].Consequently, S6K1 is a promising target for the development of treatments to alleviate peripheral nervous system damage.Tis study aims to investigate changes in these proteins in diabetic peripheral neuropathy (DPN).
Te objective of this investigation is to evaluate the efcacy of islet transplantation and sildenafl on diabetic peripheral neuropathy (DPN) both in vivo and in vitro and to explore the potential of combining these treatments as a therapy for DPN.Our hypothesis is that active islets can activate the mTOR/S6K pathway, leading to the restoration of BDNF and MBP expression in Schwann cells, ultimately resulting in the mitigation of peripheral nerve injury.Furthermore, we postulate that the inclusion of sildenafl can enhance the activity of transplanted islets, shield them from damage, and potentiate their function.

Animal Experiments.
Mature male C57BL/6 mice (8week-old, 20-25 g) were purchased from the Laboratory Animal Center (Wenzhou Medical University, Wenzhou, China).Te environment mice accommodated in were specifed pathogen free (SPF) at 24 °C ± 1 °C and on a 12-hour 2 International Journal of Endocrinology light/dark cycle for at least one week before the experiment.Ten, twenty mice, which fasted for 6-8 hours before every injection, were injected with streptozotocin (STZ, 60 mg/kg, Sigma Aldrich, St. Louis, USA) intraperitoneally for 5 consecutive days to induce diabetes (blood glucose > 16.67 mmol/L).After a 16-week feeding, the diabetic mice were randomly divided into 4 groups: DB group (n � 5, mice with diabetes), DB + sildenafl group (n � 5, DB mice intragastric administration of sildenafl, 20 mg/kg per day, Pfzer Inc., York, USA), DB + IT group (n � 5, DB mice treated with islet transplantation), and DB + IT + sildenafl group (n � 5, DB mice treated with islet transplantation and sildenafl).Tere were another fve healthy mice were marked as the control group (control).All animals were sacrifced and the tissues were collected 4 weeks after diferent treatments.

Islet Transplantation.
Mature male C57BL/6 mice used as islet donors were anesthetized with isofurane.After ligation of the ampulla of Vater, 5 mL collagenase V (Gibco, CA, USA) was injected into the pancreatic duct via the common bile duct.Te swollen pancreas was then removed and put into a 37 °C waterbath for digestion.Islets were isolated by density gradient centrifugation with Histopque-1077 and picked up into a black glass petri dish.Ten, they were cultured in RPMI-1640 (Gibco, California, USA) for 4-6 hours, and the viability of the islets was evaluated by FDA-PI (fuorescein diacetate-propidium iodide, Solarbio, Beijing, China) before surgery.After exposing the diabetes mouse's kidneys, islets equivalent to 250-350 islets (IEQ) were slowly injected into the renal capsule.

Hematoxylin-Eosin (HE) and Luxol Fast Blue Staining.
After being fxed with 4% paraformaldehyde, the renal and sciatic nerve tissues were cut into 5 μm parafn samples.For HE staining, the sections were performed as previously described [14].For Luxol fast blue staining, the sciatic nerve samples were then deparafnized and hydrated in 95% ethanol.Tey were put into Luxol fast blue solution (Solarbio, Beijing, China) for staining overnight at room temperature.95% ethanol was used to wash away excess dyes.Te sections were put into lithium carbonate and 70% ethanol, respectively.After dehydration, clearing, and mounting, the pathological changes of sciatic nerves were observed under a light microscope, and the diameter of axons and nerve fbers and the thickness of myelin sheath were measured.

Immunohistochemistry.
Te parafn renal and sciatic nerve tissues were dewaxed in xylene for 2 hours.After gradient dehydration in alcohol, the samples were boiled for 15 minutes with maximum power in citric acid-hydrochloric acid solution (0.01 mol/L).Te sections were incubated in hydrogen peroxide (3% H 2 O 2 ) for 10 minutes to reduce the efect of endogenous peroxidase and then blocked with 5% normal goat serum.After an overnight incubation with primary antibodies against insulin (Cell signal Technology Company, Boston, USA), BDNF (Afnity, USA), MBP (Proteintech, USA), and cleaved caspase-3 (Cell signal Technology Company, Boston, USA) at 4 °C, the sections were further covered with peroxidase-coupled secondary antibody for 1 hour.At last, sections were stained with diaminobenzidine (Beyotime, China).Positive areas were measured by calculating the mean integrated optical density (IOD).

Cell Viability Assay.
Cells were cultured in 96-well plates at a density of 10000 cells per well.After cultured with 5 Mm glucose (C), 100 mM glucose (HG), and 5 mM glucose + 95 mM mannitol group (mannitol) for 24 hours and 48 hours, the cell viability was detected by Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Japan).

Statistical Analysis.
Data are shown as the mean-± standard deviation (SD), analyzed with GraphPad Prism 7.0 software.All data passed tests for normality and homogeneity of variance.Student's t-test and one-way analysis of variance (ANOVA) were used for statistical analyses.Multiple comparisons between groups were analyzed using Tukey's multiple comparisons test.Statistical signifcance was accepted when P < 0.05.

Establishment of Diabetic Mouse Model, Evaluation of Donor Islets Activity, and Islet Transplantation Efect.
Following the administration of streptozotocin (STZ) injection for fve consecutive days and a normal diet for one week, blood glucose concentrations were determined in C57BL/6 mice by collecting blood from the tail vein.Te diabetic mice exhibited a signifcant increase in blood glucose levels compared with normal mice, and their body weight decreased progressively with the development of diabetes (Figure 1(a)).

International Journal of Endocrinology
Healthy male C57BL/6 mice were anesthetized, and their pancreatic tissues were extracted, digested, and subjected to gradient centrifugation to isolate high-purity islets (Figure 1(b)).After 4-6 hours of culture, some islets were randomly selected for FDA-PI fuorescence staining.Under the fuorescence microscope, the cells with good activity emitted green fuorescence and the cells with weak activity emitted red light.FDA-PI staining results showed that the extracted islet cells maintained good activity, and the proportion of active cells in islets was more than 95% (Figure 1(c)).
Six weeks following subcapsular islet transplantation (Figure 1(d)), the transplanted kidneys were removed for tissue sections.HE staining and immunohistochemical staining revealed that tissues under the renal capsule were capable of normal insulin secretion (Figure 1(e): arrow pointing to the positive site of insulin expression and secretion).
According to the records of body weight and random blood glucose of each group during the experiments, the body weight of diabetic mice began to increase gradually after islet transplantation.While the blood glucose level of the transplantation group exhibited fuctuations, it was signifcantly distinct from that of diabetic mice without islet transplantation and closely approximated the blood glucose level of normal mice.Administration of sildenafl alone showed no signs of improvement in body weight and blood glucose.protein is decreased, indicating that the nerve fber myelin sheath is damaged.In the transplantation group, the expression of cleaved caspase-3 decreased, while the expression of BDNF and MBP increased, indicating a restoration of the nerve fber myelin sheath.

Sildenafl Can Enhance the Terapeutic Efect of Islet Transplantation on Sciatic Nerve Injury in Diabetic Mice.
Based on the aforementioned experimental results, the present study elucidates that islet transplantation has the potential to ameliorate sciatic nerve injury in diabetic mice.
In addition, our fndings indicate that while the diabetic mice treated solely with sildenafl did not exhibit a notable repair efect, the combined treatment of islet transplantation and sildenafl resulted in a more apparent recovery when compared to the group treated only with islet transplantation, as demonstrated by the results in Figures 2 and 3.

Islet Cells Attenuate RSC96 Cell Injury Induced by High
Glucose by Upregulating the mTOR/S6K1 Pathway.Te present study investigated the efect of islet transplantation on RSC96 cells under high glucose condition.Results from the CCK-8 experiment demonstrated a signifcant decrease in the activity of RSC96 cells cultured in high glucose (100 mmol/L) for 48 hours (Figure 4(a)).At the same time, there was no diference in the activity between the mannitol group and the normal group, excluding the efect of osmotic pressure on cell damage.To further observe the efect of islets on RSC96 cells under high glucose condition, we cocultured the extracted islets with RSC96 cells (Figure 4(b)).Western blotting results showed that the phosphorylation levels of mTOR and S6K1 in RSC96 cells were signifcantly decreased in the high glucose group, but this phenomenon was reversed in RSC96 cells in the islets coculture group, and the degree of phosphorylation was restored (Figures 4(c) and 4(d)).Similarly, the expression of BNDF and MBP proteins decreased under high glucose and was downregulated after islet coculture (Figures 4(e) and 4(f )).
To further verify the protective efect of islet cells on this pathway, we used rapamycin (0.1 μmol/L), a potent inhibitor of the mTOR pathway.Te addition of rapamycin to the coculture system resulted in the attenuation of islets on mTOR/S6K1 pathway functionality in RSC96 cells (Figures 4(g) and 4(h)).In addition, the level expression of BDNF and MBP decreased at the same time (Figures 4(i) and 4(j)).Collectively, our fndings suggest that islet transplantation can protect RSC96 cells against high glucoseinduced damage by restoring the mTOR/S6K1 pathway and upregulating the expression of BNDF and MBP proteins.

Sildenafl Can Enhance the Terapeutic Efect of Islet Cells against RSC96 Cell Damage Induced by High Glucose.
In further experiments, sildenafl (0.1 μmol/L) was added to the system in which islets were cocultured with RSC96 cells under high glucose.Similar to the results in animal experiments, we observed that sildenafl alone had limited efect on RSC96 cells.However, upon the addition of sildenafl to the coculture system, a more pronounced mTOR/ S6K1 phosphorylation level was observed in RSC96 cells when compared to the coculture group (Figures 5(a) and 5(b)), and the expression level of BDNF was also higher at the same condition (Figures 5(c) and 5(d)).Moreover, we selected rapamycin to further verify the protective efect of sildenafl on islet cells by this pathway.Te results of WB showed that after the addition of rapamycin to the coculture system which was added sildenafl, the expression of BDNF and MBP decreased (Figures 5(e) and 5(f )).

Discussion
Te present investigation aimed to explore the role of sildenafl combined with islet transplantation in repairing Schwann cell injury in diabetic peripheral neuropathy.First of all, we established a model of diabetic mice and administered diferent intervention treatments to each group after stabilizing their diabetic state.Healthy adult mice were utilized as islet donors to be transplanted into the model mice.All diabetic mice who underwent islet transplantation exhibited a normalization of blood glucose levels and gradual weight gain, which was signifcantly diferent from diabetic mice who did not receive transplantation.In the International Journal of Endocrinology further pathological staining observation, the abnormal sciatic nerve tissue, the thinning and twisting of myelin sheath, and the increase of G ratio could be seen in the diabetic group.Tese phenomena were improved to some extent in the transplantation group, and immunohistochemical staining revealed that the expression of BDNF and MBP proteins in the sciatic nerve of the transplantation group returned nearly to normal, and the apoptotic protein cleaved caspase-3 decreased signifcantly.Furthermore, although the mice treated with sildenafl alone demonstrated normal performance in all aspects, the repair efect of nerve tissue injury was more apparent in the group that underwent transplantation combined with sildenafl treatment.In addition, we grouped RSC96 cells in the same manner of intervention and observed a signifcant decrease in the protein phosphorylation level of the mTOR/S6K1 pathway in high glucose (100 mmol/L) cultures, accompanied by downregulation of BNDF and MBP protein expression, which was consistent with previous studies.Upon further detection of protein expression in the coculture group, the phosphorylation level of the mTOR/S6K1 pathway upregulated and the expression of BDNF and MBP increased.Similar to the outcomes of animal experiments, we noticed that the restoration of abnormal expression caused by high glucose was more pronounced in cells cocultured with sildenafl and islets.
DPN is one of the earliest and most common complications in patients with type 1 and type 2 diabetes, which is estimated to occur in about half of patients with diabetes [26].Te onset of DPN is relatively hidden, and the progress of the disease is slow.A small number of patients will feel nerve pain, but the vast majority of patients have no obvious symptoms until the late stage of the disease, such as diabetic foot ulcers, gangrene, and other symptoms [27].Advanced DPN has a great impact on the life treatment of patients with diabetes and brings a heavy burden on patients and their families physically and psychologically.
Schwann cells form a myelin sheath structure and secrete neurotrophic factors to maintain the structure and function of peripheral nerves.Schwann cells also play an important role in the pathogenesis of DPN [28].Te morphological study of diabetic nerves found that there were periodic demyelination and myelin regeneration in the sural nerve, which suggested that Schwann cells were abnormal [29,30].Notably, Schwann cells secrete various neurotrophic factors, such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophic factor 3 (NT-3), which are essential for nerve survival and function.Clinical studies have found that serum levels of NGF and BDNF are signifcantly decreased in patients with DPN [31], and similar results were observed in the culture of primary Schwann cells of type I and type II diabetic mice and immortal rat Schwann cells under high glucose conditions [32][33][34].At the same time, a variety of signaling pathways in Schwann cells cultured with high glucose were abnormal, and fnally, high glucose would induce the apoptosis of  International Journal of Endocrinology Schwann cells [35].In Schwann cells treated with high glucose, the protein levels of p-mTOR and Bcl2 decreased, and the expression of these proteins returned to normal after treatment, and the apoptosis of Schwann cells also decreased [8,[35][36][37].Terefore, through the regulation of these signal pathways and the supplement of nutritional factors, the damage caused by DPN may have the potential to be repaired.
In our study, we found that islets can restore the abnormal signal of mTOR/S6K1 pathway in Schwann cells, that is, restore the level of phosphorylation, activate the pathway, and increase the expression of BDNF, which fully shows the protective efect of islet cells on Schwann cells and confrmed by animal experiments.Previous experiments have found that the axons of the sciatic nerve of STZinduced diabetic rats are atrophied and irregular, and the boundary of myelin sheath is unclear [36].Similar phenomena have been observed in the sciatic nerve of diabetic mice.What is exciting is that the islet transplant reversed these damages.Consequently, our results demonstrate that islet transplantation can counteract the pathological changes in the sciatic nerve of diabetic mice.
Islet transplantation is a promising treatment for patients who have sufered from type 1 diabetes for more than 5 years and developed severe hypoglycemia unawareness [38].It can maintain the blood glucose level of patients in a stable and reliable way and prevent hypoglycemia and large fuctuations of blood glucose more efectively than exogenous insulin injection, and the risk is lower than that of whole pancreas transplantation [10].However, several obstacles impede its widespread clinical application, including a shortage of qualifed islet donors and the technical challenge of islet purifcation [39].At the same time, as a graft, the maintenance of long-term function of transplanted islets will also be afected by many factors, including immune rejection, limited proliferation of transplanted cells, necrosis, and apoptosis [10].Terefore, it is necessary to develop strategies to improve the activity of transplanted islets or reduce their loss during transplantation.Sildenafl, a vasodilator, was initially developed for the treatment of neonatal pulmonary hypertension, but now, it is mostly mentioned because of its amazing efect in the treatment of erectile dysfunction in men.Beyond this, sildenafl has demonstrated its versatility as a potential therapeutic agent in numerous felds.Members of the PDE-5i drug family, including sildenafl, are currently being investigated as a new direction for cancer treatment [40].In the realm of transplantation, studies have revealed that sildenafl can promote the recovery of myocardial function after ischemia associated with heart transplantation [41] and aid in the success of lung, liver, and kidney transplantation [42][43][44].In addition, an in vitro study has demonstrated that sildenafl citrate can afect the immune system by reducing the production of TNF-α by T lymphocytes [45].It shows that sildenafl may have a certain immunosuppressive efect.Tis coincides with the research direction of the protection of transplanted islet cells by TNF-α inhibitors [46].
Terefore, we introduced sildenafl into the experiment of islet transplantation, hoping to explore whether sildenafl can play a positive role in islet transplantation.Te results were satisfactory and we observed that with the help of sildenafl, whether cocultured in vitro or transplanted in vivo, the protective efect of islets was magnifed to a certain extent.Tis fnding provides a new direction of thinking for maintaining the long-term and stable activity of islet cells.If those efects can be further confrmed, sildenafl can reduce the dosage of donor islets or the loss during inhibition to a certain extent in the case of lack of donor islet resources and provide the possibility of islet transplantation for more patients with diabetes and its complications.Further confrmation of these efects will be instrumental in advancing the development of new and efective therapies for this patient population.
Te present study, however, has several limitations that require further investigation.In the next work, we hope to explore the possibility of reducing the required amount of islet transplantation with sildenafl.We propose the establishment of a transplantation group combined with sildenafl, which can be compared with the normal transplantation group.If there are no signifcant diferences between the two groups, we can confrm our hypothesis.In International Journal of Endocrinology addition, for the study of DPN, because of the limitations of experimental conditions, it is a pity that we do not have relevant behavioral records in animal experiments, which we hope to be supplemented and improved in the following research.

Conclusion
Our experiments confrmed that islet transplantation signifcantly improved blood glucose and body weight of diabetic mice, restored the morphological structure of the myelin sheath of the sciatic nerve, and upregulated the expression of BDNF and MBP in the tissue.By regulating the mTOR/S6K1 pathway, islet cells can protect Schwann cells cultured in high glucose.At the same time, in animal experiments and cell experiments, it was observed that the efect of islet cells was increased under sildenafl combined therapy.Tis provides a new idea for individualized efcacy evaluation and targeted therapy in patients with diabetes mellitus and its complications.

Figure 1 :
Figure 1: Establishment of the diabetes model, evaluation of donor islet activity, and efect of islet transplantation.(a) Te body weight and blood glucose of mice in each group during the whole experiment.(b) Islets extracted from the pancreas of healthy donor mice.(c) Islet transplantation under the renal capsule (Scale � 200 μm).(d) FDA-PI staining used to detect the activity of isolated islets during culture (scale � 200 μm).(e) Te results of HE staining (A) and insulin antibody immunohistochemical staining (B) of the kidney after islet transplantation in mice (immunohistochemical staining: ×200).

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International Journal of Endocrinology3.2.Islet Transplantation Reduces the Pathological Injury of the Sciatic Nerve in Diabetic Mice.Trough the results of HE staining of sciatic nerve sections in mice, we can observe the atrophy and distortion of sciatic nerve tissue in diabetic mice.After islet transplantation, most of the sciatic nerve morphology returned to normal (Figure2(a)).Myelin structure is an important part of nerve fbers.We stained the myelin sheath of the sciatic nerve of mice by fast blue staining and measured the myelin sheath thickness (Figure2(c)).It was found that the myelin sheath became thinner in the diabetic group (Figure2(b)).By measuring the diameter of axons and nerve fbers, calculating the ratio (G ratio), it was found that the G ratio of diabetic mice increased (Figure2(d)).Te G ratio is related to the state of nerve fbers, especially the conduction of nerve signals, which further indicates the decrease of the thickness of the myelin sheath and the damage of myelin sheath.After islet transplantation, the injured myelin sheath recovered, the thickness increased, and the G ratio decreased.3.3.Islet Transplantation Restored the Expression of Nutritional Factors and Reduced the Expression of Apoptotic Proteins in the SciaticNerve of Diabetic Mice.Te expression of structural and nutritional factors and apoptotic proteins in the sciatic nerve tissue of mice in each group was detected by immunohistochemical staining to further evaluate the state of nerve fbers in each group.According to the semiquantitative analysis of the expression of each protein, we can observe that compared with the normal group, the expression of apoptotic protein cleaved caspase-3 in the sciatic nerve of diabetic mice is signifcantly increased (Figures3(a) and 3(d)), while the expression of BNDF (Figures 3(b) and 3(e)) and MBP (Figures 3(c) and 3(f ))

Figure 2 :
Figure 2: Pathological staining, myelin thickness, and G value of sciatic nerve sections in each group.(a) HE staining of sciatic nerve tissue of mice in each group.(b) Te thickness of sciatic nerve fber myelin sheath in each group.(c) Fast blue staining of the myelin sheath of the sciatic nerve of each group (n � 3).Te arrow points to a thinned and twisted myelin sheath in diabetic mice.(d) G value of mice in each group.* P < 0.05, * * * * P < 0.0001.

Figure 4 :
Figure 4: Efects of islets on mTOR/S6K1 phosphorylation and expression of BDNF and MBP proteins in RSC96 cells under high glucose conditions.(a) Te results of viability assay (CCK-8) of RSC96 cells cultured in high glucose for 24 and 48 hours.(b) Pancreatic islets were cocultured with RSC96 cells.(c,d) Te level and semiquantitative analysis of total protein and phosphorylated protein of mTOR/S6K1 in RSC96 cells cocultured with islets under high glucose condition.(e, f ) Te level and semiquantitative analysis of BDNF and MBP proteins in RSC96 cells cocultured with islets under high glucose condition.(g, h) Te analysis of total protein and phosphorylated protein of mTOR/ S6K1 in RSC96 cells cocultured with islets following rapamycin pretreatment under high glucose condition.(i, j) Te analysis of BDNF and MBP proteins in RSC96 cells cocultured with islets following rapamycin pretreatment under high glucose condition.* P < 0.05, * * P < 0.01, * * * P < 0.001, and * * * * P < 0.0001.

Figure 5 :
Figure 5: Efects of sildenafl combined with islets on mTOR/S6K1 phosphorylation and expression of BDNF and MBP proteins in RSC96 cells under high glucose conditions.(a, b) Te level and semiquantitative analysis of mTOR/S6K1 total protein and phosphorylated protein in RSC96 cells cocultured with sildenafl and islets under high glucose condition.(c, d) Te level and semiquantitative analysis of BDNF and MBP proteins in RSC96 cells cocultured with sildenafl and islets under the condition of high glucose.(e, f ) Te analysis of BDNF and MBP proteins in RSC96 cells cocultured with sildenafl and islets following rapamycin pretreatment under high glucose condition.* P < 0.05, * * P < 0.01, * * * P < 0.001, and * * * * P < 0.0001.