Discussion on Therapeutic Effect of ALA on Diabetic Peripheral Neuropathy rats from mitochondrial transport

Discussion on Therapeutic Effect of ALA on Diabetic Peripheral Neuropathy rats from mitochondrial transport | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Discussion on Therapeutic Effect of ALA on Diabetic Peripheral Neuropathy rats from mitochondrial transport Jiaxin Tian, jingwen An, Linchun Song, Wang Zhang, Die Chen, Tianya Zhang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3687044/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose To investigate the AMPK/CREB pathway–mediated effect of alpha-lipoic acid (ALA) on the sciatic nerve of rats with diabetic peripheral neuropathy (DPN) and to attempt to elucidate the underlying mechanism. Methods In vivo experiment, healthy male Sprague-Dawley (SD) rats were induced by high-carbohydrate/high-fat diet and intraperitoneal injection of streptozotocin (STZ) (30 mg·kg − 1 ) to induce diabetes. The diabetes SD rats were randomly divided into DPN group and alpha lipoic acid (ALA) group (n = 15). The other 15 SD rats were set as Control group. Then Control group and DPN group received the same amount of normal saline by intragastric administration, and ALA groups received ALA intervention every day for 12 weeks. Motor nerve conduction velocity (MNCV) and Paw Withdrawal Threshold (PWT) were detected. The morphological changes were observed by HE staining in sciatic nerves. Kinesin family member 5A (KIF5A), Dynein Cytoplasmic 1 Intermediate Chain 2 (DYNC1I2), phosphorylated Adenosine 5'-monophosphate (AMP) activated protein kinase (p-AMPK), Adenosine 5'-monophosphate (AMP) activated protein kinase (AMPK), phosphorylated cAMP responsive element binding protein(p-CREB) and cAMP responsive element binding protein(CREB)were observed by immunofluorescence assay and Western blot. In the vitro cell experiment, the NSC34 cells injury model was established by treating with 50 mmol·L − 1 of high glucose and 250 µmol·L − 1 of palmitic acid sodium. NSC34 cells were randomly divided into Control group, Model group and alpha lipoic acid intervention group (ALA group). ALA group was given corresponding ALA for 24 hours of intervention. The axon changes were observed by measured neuron axon length in NSC34 cells. KIF5A, DYNC1I2, p-AMPK,AMPK, p-CREB and CREB were observed by immunofluorescence assay and Western blot. Results ALA improved the MNCV and PTW of rats with DPN and reduced their mechanical pain threshold. AMPK was activated by ALA. p-CREB, KIF5A expression was upregulated, while DYNC1I2 expression was downregulated. ALA regulates mitochondrial transport in peripheral nerve. Conclusion ALA activates CREB and KIF5A through AMPK, regulates positive mitochondrial transport, protects axons, and attenuates DPN. Alpha-lipoic acid Diabetic peripheral neuropathy Sciatic nerve AMPK Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Diabetic peripheral neuropathy (DPN) is the most prevalent complication of diabetes mellitus (DM) and a major cause of disability. The most common form of DPN is distal symmetrical multiple peripheral neuropathy, which primarily affects sensory, motor, and autonomic nerves 1 . In severe cases, DPN can result in intense pain and even require amputation, significantly impacting the quality of life for patients 2 . Currently, there is no approved therapy or medication specifically for DPN. Peripheral nerve demyelination and axonal degeneration are the primary pathological characteristics of DPN 3 . The survival of peripheral nerve axon ends heavily relies on energy support. Due to the neuron is a site of high energy demand and its unique polarized structure, it requires mitochondrial transport to maintain energy homeostasis throughout the cell, especially in the distal axon 4 , 5 . To maintain axonal bioenergy, neurons deploy complex transport mechanisms that transport healthy mitochondria as a local energy source to the terminal and remove damaged mitochondria from the distal region 6 . Neurons maintain energy homeostasis in axons through mitochondrial transport. One of the motor proteins involved in the anterograde axonal mitochondrial transport is Kinesin family member 5A (KIF5A), which is crucial for maintaining the vitality and function of neurons 7 . Dynein is a crucial protein responsible for the reverse transport of mitochondria in axons of neurons. Its main function is to timely transport aging or damaged mitochondria at the end of axons back to the neuron cell body 8 . Dynein cytoplasmic 1 intermediate chain 2 (DYNC1I2) serves as the intermediate chain of Dynein. The transport of axon mitochondria is regulated in order to respond to changes in the biological energy status as neurons grow and become damaged, ensuring an adequate supply of ATP at these metabolically active locations. AMPK plays a crucial role in maintaining cell energy balance and has been found to be involved in various physiological activities 9 . Activation of AMPK can lead to the activation of CREB and enhance its transcription. CREB, as an important nuclear transcription factor, can upregulate KIF5A, thereby promoting forward mitochondrial transport 10 .It has been observed that dysregulation of AMPK signaling and impaired axon energy metabolism are present in certain neurodegenerative diseases 11 , 12 . However, there has been limited studies investigating the impact of AMPK on the sciatic nerve in the development of DPN. DPN is a type of injury that affects nerves in a length-dependent manner, with early neuronal dysfunction potentially occurring in the distal axon. This suggests that the mechanism of mitochondrial transport plays a significant role in delivering mitochondria from the cell body to the axon for proper neuronal functioning. ALA, a derived antioxidant synthesized in mitochondria, serves as a cofactor 13 . It exhibits a potent antioxidant effect and is frequently used in the prevention and treatment of DPN. ALA mediates the activation of AMPK by serine/threonine kinase (LKB1) or calmodulin-dependent protein kinase (CAMKK) 14 .Can ALA regulate mitochondrial axon transport and protect axons in diabetes by activating AMPK? The following experimental studies were carried out. 2. Materials and methods 2.1.1 Animals The animal experiment was approved by the Guiding Prin-ciples for the Care and Use of Laboratory Animals published by the National Science and Technology Commission of China. Forty-five male Sprague-Dawley rats (7–8 weeks old, 200–240 g) were purchased from Liaoning Changsheng Biotechnology Co. LTD. (Beijing, China) (SCXK (Liaoning) 2020-0001) and housed in 15 cages in a facility with a 12-h/12-h artificial light–dark cycle, a temperature of 22–24°C, and a humidity level of 40%. The cage bedding was changed daily. 2.1.2 Induction of diabetes mellitus and drug treatment Fifteen rats were randomly assigned to the control group and fed a standard diet. The remaining rats were fed a high-carbohydrate/high-fat diet for 6 weeks and were then intraperitoneally injected with STZ (Sigma-Aldrich, St Louis, MO, USA) (30 mg/kg). After 1 week, rats with a fasting glucose level ≥ 16.7 mmol/L were considered to be diabetic and continued to be fed a high-carbohydrate/high-fat diet. The diabetic rats were randomly divided into a DPN group and an ALA group, with 15 rats in each group. The rats in the ALA group were intragastrically administered an ALA suspension (60 mg/kg/day) for 12 weeks. The rats in the control group and DPN group were intragastrically administered an equal volume of distilled water every day for 12 weeks. After the treatment, the motor nerve conduction velocity and mechanical pain threshold of the diabetic rats in the ALA group and the DPN group were observed to be reduced to varying degrees: the rats in both the ALA group and the DPN group were therefore considered to have DPN 15 . 2.1.3 Blood glucose level and weight analysis The diabetic rats having been identified, fasting glucose levels and body weight were measured and recorded every 4 weeks. 2.1.4 Mechanical pain threshold measurement The mechanical pain threshold was measured by using an electronic von Frey (type 2390, 90 g probe, 0.8 mm diameter; IITC Life Science Inc. Woodland Hills, CA, USA) after the 12-week treatment period. The rats were placed on wire mesh, covered with a glass box, and allowed to acclimate to their surroundings for 15 min. A stainless-steel filament was applied vertically to the plantar surface of the hind paw. Rapid paw withdrawal by the rats was considered to be a positive response, and the force that elicited the response was recorded (grams). Paw withdrawal caused by the physical activity of the rats was considered to be a negative response. Measurements were taken at intervals of 3–5 min. The response of each paw was assessed three times. 2.1.5 Motor Nerve conduction velocity After the 12-week treatment period, the MNCV of the rats was measured. The rats were anesthetized with isoflurane (3% volume) before being fixed in the prone position. The sciatic nerve was exposed through an incision, and the skin between the biceps femoris and semitendinous muscles was separated from the muscles on the experimental side. Electrodes were implanted in two sites of the sciatic nerve notch approximately 10 mm apart. Platinum wire electrodes were placed directly under the sciatic nerve trunk in the right leg for stimulation, after which the data were recorded. The sciatic nerve was stimulated with a single square-wave pulse (intensity: 1.2 V, width: 1 ms) using an experimental system (BL-420s, Taimeng, Sichuan, China). The MNCV was measured and calculated as follows: MNCV = D/L (m/s). 2.1.6 Immunofluorescence assay For fluorescence staining, sciatic nerve and dorsal root ganglions sections underwent routine deparaffinization and rehydration and were then subjected to antigen retrieval by incubation with citric acid (0.01 mol/L, pH 6.0) in a microwave oven at approximately 90°C for 20 min. Next, the sections were blocked with non-immunoreactive serum and then incubated with antibodies against KIF5A (Abcam,USA,1:500), DYNC1I2 (Proteintech,USA,1:100), p-AMPK(Immunoway,USA,1:100), p-CREB(Abcam,USA,1:100) at 4°C overnight and subsequently incubated with secondary antibodies at 37°C for 1 h. After being stained, sections were incubated with DAPI for nuclear staining and sealed for photography using an inverted fluorescence microscope (CTS SP8, Leica, Germany). 2.1.7 Western blotting for the detection of related proteins in tissues Sciatic nerve and dorsal root ganglion tissues were homogenized in RIPA buffer, and protein samples were separated by 10% SDS–PAGE and transferred onto nitrocellulose membranes (Pall Gelman, Ann Arbor, MI, USA). After transfer, the membranes were blocked with 5% skim milk powder. Then, the membranes were incubated with antibodies against KIF5A(Abcam,USA,1:1000), DYNC1I2(Proteintech,USA,1:500), AMPK(Immunoway,USA,1:1000), p-AMPK(Immunoway,USA,1:1000), CREB(Proteintech,USA,1:500), p-CREB(Abcam,USA,1:500) at 4°C overnight followed by a fluorescent secondary antibody for 1 h at room temperature. A laser infrared scanner (Odyssey, LI-COR, USA) was used to visualize the protein bands. 2.2.1 Cell culture assays in vitro NSC34 cells were maintained in culture media with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin and streptomycin at 37°C in an incubator with a humidified atmosphere and 5% CO2. NSC34 cells were divided into the Control group, DPN group, ALA group. Except the Control group, the other groups were added 50mmol·L − 1 glucose and 250µmol·L − 1 palmitic acid. ALA group were given 250µM ALA 16 . Relevant indexes were detected 24h after administration. 2.2.2 Immunofluorescence analysis The dried and sterilized glass slides were placed on a 24-well petri dish, the cells were spread evenly on the glass slides in the wells, and 4% paraformaldehyde was added and incubated for 20 min at room temperature. Then 0.25% Triton X-100 was added and incubated for 15 min for permeabilization, and 10% normal goat serum was added and incubated for 30 min for blocking. Subsequently, the cells were incubated with the primary antibody KIF5A(Abcam,USA,1:1000), DYNC1I2(Proteintech,USA,1:500), AMPK(Immunoway,USA,1:1000), p-AMPK(Immunoway,USA,1:1000), CREB(Proteintech,USA,1:500), p-CREB(Abcam,USA,1:500) overnight at 4°C, followed by the relevant secondary antibody at 37°C in the dark for 1 h for fluorescence staining. DAPI was used for nuclear staining. β-tubulin Ⅲ was used to measure and characterize the length of neurons and axons. After immunofluorescence staining, 15 different neurons were randomly selected from each group of cells, and the axonal length of the neurons was detected using Image J. 2.2.3 Western blotting for the detection of related proteins in cells NSC34C cells were homogenized in RIPA buffer, and protein samples were separated by 10% SDS–PAGE and transferred onto nitrocellulose membranes (Pall Gelman, Ann Arbor, MI, USA). After transfer, the membranes were blocked with 5% skim milk powder. Then, the membranes were incubated with antibodies against KIF5A(Abcam,USA,1:1000), DYNC1I2(Proteintech,USA,1:500), AMPK(Immunoway,USA,1:1000), p-AMPK(Immunoway,USA,1:1000), CREB(Proteintech,USA,1:500), p-CREB(Abcam,USA,1:500) at 4°C overnight followed by a fluorescent secondary antibody for 1 h at room temperature. A laser infrared scanner (Odyssey, LI-COR, USA) was used to visualize the protein bands. 2.3 Statistical analysis SPSS 24.0 (IBM Corp. Released 2016. IBM SPSS Statistics for Windows, Version 24.0. Armonk, NY: IBM Corp) was used to analyze the data. All data are presented as mean ± standard deviation (M ± SD). Normally distributed data were analyzed with one-way ANOVA with Tukey’s test for multigroup independent samples. Statistical tests were performed using two-sided tests. P < 0.05 was considered significant. 3. Results 3.1 Body weight and fasting blood glucose levels Table 1 Effects of ALA on body weight in rats (g) Groups 0 Weeks 4 Weeks 8 Weeks 12 Weeks Control 330.38 ± 9.05 389.91 ± 13.30 405.83 ± 14.03 412.54 ± 13.98 DPN 356.36 ± 18.95 415.29 ± 10.94 416.29 ± 17.63 414.54 ± 14.03 ALA 332.87 ± 13.26 407.48 ± 16.32 413.49 ± 8.05 420.86 ± 15.61 Values are presented as mean ± standard deviation, n = 15 for each group DPN diabetic peripheral neuropathy, ALA α-lipoic acid. Table 2 Effects of ALA on fasting blood glucose in rats (mmol/L) Groups 0 Weeks 4 Weeks 8 Weeks 12 Weeks Control 4.07 ± 0.52 4.55 ± 0.43 4.43 ± 0.46 4.89 ± 0.46 DPN 26.26 ± 2.53 ## 28.56 ± 2.49 ## 28.92 ± 2.54 ## 26.70 ± 4.55 ## ALA 27.43 ± 3.93 ## 28.27 ± 2.34 ## 26.91 ± 2.06 ## 26.71 ± 2.56 ## Values are presented as mean ± standard deviation, n = 15 for each group # # P < 0.01 vs. the control group. One-way ANOVA with Tukey’s multiple comparisons test. As shown in Table 1 , the body weight of the rats that were fed a high-carbohydrate/high-fat diet was significantly increased after 4 weeks. As shown in Table 2 , fasting blood glucose levels were significantly higher in both DPN and ALA group compared with Control group. Compared with DPN group, there was no significant difference in fasting blood glucose in ALA group. Previous studies have shown that reducing blood glucose levels does not slow down the progression of the disease Although ALA has a mild hypoglycemic effect, our experiment found no significant impact of ALA on reducing blood glucose levels in rats. Therefore, the therapeutic effect of ALA on DPN was not achieved through the reduction of blood glucose levels. 3.2 Pain thresholds Table 3 Effects of ALA on MNCV, the paw withdrawal threshold in rats Groups PTW(g) MNCV(m/s) Control 79.84 ± 5.62 53.53 ± 3.28 DPN 23.92 ± 6.26 ## 29.84 ± 2.35 ## ALA 50.93 ± 7.85 ** 38.78 ± 2.83 ** Values are presented as mean ± standard deviation, n = 15 for each group MNCV motor nerve conduction velocity ## P < 0.01 vs. the control group; ** P < 0.01 vs. the DPN group. One-way ANOVA with Tukey’s multiple comparisons test. As illustrated in Table 3 , the rats in the DPN group were more sensitive to mechanical stimulation than the normal rats (P < 0.01); however, the mechanical thresholds for paw withdrawal of the ALA-treated rats were markedly increased compared with those of the rats in the DPN group(P < 0.01). 3.3 MNCV As shown in Table 3 , the MNCV of the DPN group was clearly lower than those of the control group (P < 0.01), and the MNCV of the ALA group were significantly higher than those of the DPN group (P < 0.01). 3.4 Pathological changes of sciatic nerves(HE) As shown in Fig. 1 , HE staining microscopy showed that the structure of nerve fibers in the sciatic nerve in the Control group was normal, without swelling or atrophy of axons, and the structure of myelin sheath was complete. In DPN group, there was a large amount of swelling of nerve axons, partial atrophy, and large loss of myelin sheath. The axonal swelling and demyelination in ALA group was less, and axonal atrophy was not obvious. 3.5 Immunofluorescence and western blot results in vivo As shown in Fig. 2 a, b, c, the expression of KIF5A in the DPN group was decreased compared with that in the control group (P < 0.01). Additionally, KIF5A levels were increased in the ALA group compared with the DPN group (P < 0.01). These results indicate that ALA can increase the level of anterograde mitochondrial transport in rats with DPN. The trends revealed by western blotting were consistent with the expression data obtained by immunofluorescence. As shown in Fig. 3a, b, c, the expression of DYNC1I2 in the DPN group was increased compared with that in the control group(P < 0.01). Additionally, DYNC1I2 (P < 0.05) levels were decreased in the ALA group compared with the DPN group. These results indicate that ALA can decrease the level of retrograde mitochondrial transport in rats with DPN. The trends revealed by western blotting were consistent with the expression data obtained by immunofluorescence. As shown in Fig. 4 a, b, c,the expression of p-AMPK/AMPK in the DPN group was decreased compared with that in the control group(P < 0.01). Additionally, the p-AMPK/AMPK levels were increased in the ALA group compared with the DPN group(P < 0.05). These results indicate that ALA can increase the level of p-AMPK in rats with DPN. The trends revealed by western blotting were consistent with the expression data obtained by immunofluorescence. As shown in Fig. 5 a, b, c, the expression of p-CREB/CREB in the DPN group was decreased compared with that in the control group (P < 0.05). Additionally, the p-CREB/CREB levels were increased in the ALA group compared with the DPN group (P < 0.01). These results indicate that ALA can increase the level of p-CREB, which is a downstream factor of p-AMPK, in rats with DPN. The trends revealed by western blotting were consistent with the expression data obtained by immunofluorescence. 3.6 Immunofluorescence and western blot results in vitro As shown in Fig. 6 a, b, the neuron axon length in the Model group was decreased compared with that in the control group(P < 0.01). Additionally, neuron axon length levels were increased in the ALA group compared with the Model group (P < 0.01). These results indicate that ALA can increase the axon length in Model group of NSC34 cells. As shown in Fig. 7a, b, c,the expression of KIF5A in the Model group was decreased compared with that in the control group(P < 0.01). Additionally, KIF5A levels were increased in the ALA group compared with the Model group (P < 0.05). These results indicate that ALA can increase the level of anterograde mitochondrial transport in Model group of NSC34 cells. The trends revealed by western blotting were consistent with the expression data obtained by immunofluorescence. As shown in Fig. 8 a, b, c, the expression of DYNC1I2 in the Model group was increased compared with that in the control group (P < 0.01). Additionally, DYNC1I2 levels were decreased in the ALA group compared with the Model group (P < 0.01). These results indicate that ALA can decrease the level of retrograde mitochondrial transport in Model group of NSC34 cells. The trends revealed by western blotting were consistent with the expression data obtained by immunofluorescence. As shown in Fig. 9 a, b, c, the expression of p-AMPK/AMPK in the Model group was decreased compared with that in the control group (P < 0.01). Additionally, the p-AMPK levels were increased in the ALA group compared with the Model group (P < 0.01). These results indicate that ALA can increase the level of p-AMPK in Model group of NSC34 cells. The trends revealed by western blotting were consistent with the expression data obtained by immunofluorescence. As shown in Fig. 10 a, b, c, the expression of p-CREB in the Model group was decreased compared with that in the control group (P < 0.01). Additionally, the p-CREB levels were increased in the ALA group compared with the Model group (P < 0.01). These results indicate that ALA can increase the level of p-CREB, which is a downstream factor of p-AMPK, in Model group of NSC34 cells. The trends revealed by western blotting were consistent with the expression data obtained by immunofluorescence. 4. Discussion The mechanism of DPN is not fully understood and treatments are still being explored. ALA is a potent antioxidant that can effectively eliminate various reactive oxygen species. This effect has been applied to the prevention and treatment of DPN 22 . But we overlooked another aspect of ALA's efficacy: AMPK agonists. The purpose of this study was to investigate the protective effect of ALA on peripheral nerves in DPN rats based on AMPK activation. Our results suggest that ALA can regulate the CREB/KIF5A pathway by activating AMPK, improve mitochondrial forward transport, protect sciatic nerve axon terminus, and prevent DPN. AMPK is a cellular energy sensor expressed by a variety of organs and is closely related to metabolic diseases such as cancer, obesity and diabetes 23 . The role of AMPK pathway in the prevention and treatment of diabetic complications has received increasing attention in recent years. CREB is the downstream target of AMPK, and as a crucial nuclear transcription factor, it can increase the level of kinase protein subunits when activated 24 . Numerous studies have demonstrated a synergistic relationship between AMPK and CREB and their involvement in adaptive responses to cellular energy stress. In the AMPK/CREB metabolic control network, p-AMPK can activate CREB, thereby regulating the expression of the anterograde axon mitochondrial transport protein KIF5A 6 , 10 . Our results show that in DPN rats, AMPK activation is decreased, CREB activation is down-regulated, and the expression of mitochondrial anterograde transport protein KIF5A is reduced, resulting in insufficient axon energy supply and neurological dysfunction. The expression of mitochondrial retrograde transport protein DYNC1I2 increased in DPN rats, which may be related to the increase of mitochondrial damage caused by high sugar. At present, the relationship between AMPK activation and DYNC1I2 expression has not been reported, which can be further explored in future studies. In this study, p-AMPK/AMPK ratio and p-CREB/CREB and KIF5A levels were up-regulated in ALA group. This suggests that ALA activates AMPK, activates its downstream target CREB, enhances the expression of KIF5A, improves the energy supply of terminal axons, and alleviates peripheral nerve injury in DPN rats. The downregulation of DYNC1I2 expression may be related to the reduction of mitochondrial damage, which is caused by the increased energy supply of terminal axons and the antioxidant effect of ALA. The peripheral neuroprotective effect of ALA is related to its regulation of mitochondrial transport at the end of axons. In vitro experiments, NSC34 cells were cultured in an environment high in sugar and fat. The NSC34 cell line is characterized by longer axons and is able to respond more significantly to axon-related damage and changes in mitochondrial transport. The results of in vitro experiments are consistent with those of animal experiments, suggesting that ALA may regulate the anteroonal transport of axon mitochondria through AMPK/CREB pathway, enhance the energy supply at the end of axon, and ultimately play a neuroprotective role. However, it is important to interpret the experimental results with caution when extrapolating treatment for diabetic peripheral neuropathy in humans because of the inevitable physiological and anatomical differences between rats and humans. In summary, our study suggests that ALA regulates mitochondrial transport by activating AMPK, effectively alleviating DPN-related symptoms. These findings provide preliminary evidence for the pharmacological effect of ALA on DPN. 5. Conclusion The findings of this study suggest that ALA has the potential to protect against DPN by regulating mitochondrial transport and improving peripheral nerve function in rats. These effects are thought to be mediated by AMPK. It is possible that ALA may also have a similar mechanism of action in the human sciatic nerve. Declarations Author Contribution Jiaxin Tian, Jingwen An Literature search and data collection, investigation, data collation, analysis, and writing-first draft, compilation of all figures. Linchun Song, WangZhang, DieChen, Tianya Zhang Search and data collection, analysis, and writing-first draft. Ying Ben review and editing. Funding This work was supported by the National Natural Science Foundation [grant numbers 81803922] funded by CHINA. Ethics approval and consent to participate This project was approved by the Ethics Committee of Hebei University of Traditional Chinese Medicine (DWLL2018042). Consent for publication Informed consent was obtained from all individual participants included in the study. Conflict of interest The authors declare no competing interests. References Zherebitskaya E, Akude E, Smith DR, Fernyhough P (2009) Development of selective axonopathy in adult sensory neurons isolated from diabetic rats: role of glucose-induced oxidative stress. Diabetes 58(6):1356–1364 Yu Y (2021) Gold Standard for Diagnosis of DPN. 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Nutrients 15 (1) Papanas N, Ziegler D (2014) Efficacy of α-lipoic acid in diabetic neuropathy. Expert Opin Pharmacother 15(18):2721–2731 Steinberg GR, Hardie DG (2023) New insights into activation and function of the AMPK. Nat Rev Mol Cell Biol 24(4):255–272 Puthanveettil SV, Monje FJ, Miniaci MC, Choi YB, Karl KA, Khandros E, Gawinowicz MA, Sheetz MP, Kandel ER (2008) A new component in synaptic plasticity: upregulation of kinesin in the neurons of the gill-withdrawal reflex. Cell 135(5):960–973 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3687044","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":255067713,"identity":"480673c3-88ba-43c3-88a5-8177980fe225","order_by":0,"name":"Jiaxin Tian","email":"","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiaxin","middleName":"","lastName":"Tian","suffix":""},{"id":255067714,"identity":"71b6d5cf-23e1-47fb-8a0e-c8b3899081d2","order_by":1,"name":"jingwen An","email":"","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"jingwen","middleName":"","lastName":"An","suffix":""},{"id":255067715,"identity":"58e3932d-7ac8-4731-9211-dededc45967b","order_by":2,"name":"Linchun Song","email":"","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Linchun","middleName":"","lastName":"Song","suffix":""},{"id":255067716,"identity":"04933c87-350f-4844-b144-ebc2a2078d2c","order_by":3,"name":"Wang Zhang","email":"","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wang","middleName":"","lastName":"Zhang","suffix":""},{"id":255067717,"identity":"96db6245-2ba5-4a14-a617-7ee70eb6b5e2","order_by":4,"name":"Die Chen","email":"","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Die","middleName":"","lastName":"Chen","suffix":""},{"id":255067718,"identity":"49a7cb22-4334-4968-98b6-bc979c71187c","order_by":5,"name":"Tianya Zhang","email":"","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tianya","middleName":"","lastName":"Zhang","suffix":""},{"id":255067719,"identity":"0b46cc5a-cbf9-40c4-bebe-6df72cb5bc6e","order_by":6,"name":"Ying Ben","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACfvaGhAMJBjX1/OzNB4jTItlz4OGBBxXHEiR7jiUQp8XgRuLjgw/OMCcYzMgxINJlN5ITDiS2seUZ8Jz5eOMNg52cbgMBHYw9z0BaZIrN2Xs3W85hSDY2O0BACzN7DtgWxp09Z7dJ8zAcSNxGSAsbQ/4HoBZmxg03cp4Rp4WHIwEYyGeYE4Fa2IjTIsFzAKil4pgxMJCNLecYEOEX++MNyR9/GNTIAaPy4Y03FXZyBLWgWUls1CBpIVXHKBgFo2AUjAgAADhTTXx9CBANAAAAAElFTkSuQmCC","orcid":"","institution":"Hebei University of Chinese Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Ben","suffix":""}],"badges":[],"createdAt":"2023-11-30 13:14:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3687044/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3687044/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":47589437,"identity":"1571ed8e-5e54-46c0-86f5-a023368c3cb8","added_by":"auto","created_at":"2023-12-04 21:42:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":173122,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ALA on morphologic changes of sciatic nerve in rats\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3687044/v1/7ebb238e95e97412f9884ea0.png"},{"id":47587951,"identity":"4c7a4d19-1dd0-4d2f-9e2d-6e24ae8afa17","added_by":"auto","created_at":"2023-12-04 21:26:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":358934,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of KIF5A expression by immunofluorescence assay and western blotting (n = 4). a Analysis of KIF5A expression by immunofluorescence assay (scale bar: 50 μm). b Expression of KIF5A determined by western blotting. c Bar plot (mean + SD) of western blot data showing KIF5A expression. ## P \u0026lt; 0.01 vs. the control group; ** P \u0026lt; 0.01 vs. the DPN group. One-way ANOVA with Tukey's multiple comparisons test\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3687044/v1/e74d0e5b10e42fb6f5b5c158.png"},{"id":47587952,"identity":"931566c6-a993-4fb1-b618-26138cea42d5","added_by":"auto","created_at":"2023-12-04 21:26:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":342425,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of DYNC1I2 expression by immunofluorescence assay and western blotting (n = 4). a Analysis of DYNC1I2 expression by immunofluorescence assay (scale bar: 50 μm). b Expression of DYNC1I2 determined by western blotting. c Bar plot (mean + SD) of western blot data showing DYNC1I2 expression. ## P \u0026lt; 0.01 vs. the control group; * P \u0026lt; 0.05 vs. the DPN group. One-way ANOVA with Tukey's multiple comparisons test\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3687044/v1/0689412ee644b97ac94c3827.png"},{"id":47587953,"identity":"61f8b162-0e39-4c4d-bc80-089ed84353c0","added_by":"auto","created_at":"2023-12-04 21:26:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":295755,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of p-AMPK expression by immunofluorescence assay and western blotting (n = 4). a Analysis of p-AMPK expression by immunofluorescence assay (scale bar: 50 μm). b Expression of p-AMPK and AMPK determined by western blotting. \u0026nbsp;c Bar plot (mean + SD) of western blot data showing p-AMPK/AMPK expression. ##P \u0026lt; 0.01 vs. the control group; *P \u0026lt; 0.05 vs. the DPN group. One-way ANOVA with Tukey's multiple comparisons test\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3687044/v1/ff9c3bbdb05d87366326d46d.png"},{"id":47589986,"identity":"4827ac4e-eada-4e7b-8a45-043f99644320","added_by":"auto","created_at":"2023-12-04 21:50:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":417807,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of p-CREB expression by immunofluorescence assay and western blotting (n = 4). a Analysis of p-CREB expression by immunofluorescence assay (scale bar: 50 μm). b Expression of p-CREB and CREB determined by western blotting. \u0026nbsp;c Bar plot (mean + SD) of western blot data showing p-CREB/CREB expression. #P \u0026lt; 0.05 vs. the control group; ** P \u0026lt; 0.01 vs. the DPN group. One-way ANOVA with Tukey's multiple comparisons test\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3687044/v1/192984885234b310c9739cea.png"},{"id":47587958,"identity":"d65da4db-7357-4381-8b42-ddc8a25c4dea","added_by":"auto","created_at":"2023-12-04 21:26:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":141832,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ALA on neuron axon length changes in NSC34 cells. a Analysis of neuron axon length by immunofluorescence assay (scale bar: 50 μm). b Bar plot (mean + SD) of immunofluorescence assay data showing neuron axon length changes. ## P \u0026lt; 0.01 vs. the control group; ** P \u0026lt; 0.01 vs. the Model group. One-way ANOVA with Tukey's multiple comparisons test\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3687044/v1/f741fb2c69de2d9991a25e9d.png"},{"id":47587954,"identity":"49a21da1-bfbe-4b72-9825-09dbd0d7707c","added_by":"auto","created_at":"2023-12-04 21:26:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":172410,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of KIF5A expression by immunofluorescence assay and western blotting (n = 4). a Analysis of KIF5A expression by immunofluorescence assay (scale bar: 50 μm). b Expression of KIF5A determined by western blotting. c Bar plot (mean + SD) of western blot data showing KIF5A expression. ## P \u0026lt; 0.01 vs. the control group; *P \u0026lt; 0.05 vs. the Model group. One-way ANOVA with Tukey's multiple comparisons test\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3687044/v1/1a6f1132959e1800dc6621f8.png"},{"id":47589439,"identity":"7fe76297-d1ad-48b7-a9a5-b8e7b2e45ee9","added_by":"auto","created_at":"2023-12-04 21:42:53","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":166198,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of DYNC1I2 expression by immunofluorescence assay and western blotting (n = 4). a Analysis of DYNC1I2 expression by immunofluorescence assay (scale bar: 50 μm). b Expression of DYNC1I2 determined by western blotting. c Bar plot (mean + SD) of western blot data showing DYNC1I2 expression. ##P \u0026lt; 0.01 vs. the control group; **P \u0026lt; 0.01 vs. the Model group. One-way ANOVA with Tukey's multiple comparisons test\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3687044/v1/3a33ff82782f2fc19143a083.png"},{"id":47587961,"identity":"df9f879d-d1c8-4b02-bccb-820795749c26","added_by":"auto","created_at":"2023-12-04 21:26:53","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":159666,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of p-AMPK expression by immunofluorescence assay and western blotting (n = 4). a Analysis of p-AMPK expression by immunofluorescence assay (scale bar: 50 μm). b Expression of p-AMPK and AMPK determined by western blotting. C Bar plot (mean + SD) of western blot data showing p-AMPK/AMPK expression. ##P \u0026lt; 0.01 vs. the control group; **P \u0026lt; 0.01 vs. the Model group. One-way ANOVA with Tukey's multiple comparisons test\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-3687044/v1/8a3d0f2abd712582dbb9208e.png"},{"id":47588901,"identity":"35894853-7bad-4bb5-a07b-3ddda13baf58","added_by":"auto","created_at":"2023-12-04 21:34:53","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":158033,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of p-CREB expression by immunofluorescence assay and western blotting (n = 4). a Analysis of p-CREB expression by immunofluorescence assay (scale bar: 50 μm). b Expression of p-CREB and CREB determined by western blotting. c Bar plot (mean + SD) of western blot data showing p-CREB/CREB expression. ##P \u0026lt; 0.01 vs. the control group; **P \u0026lt; 0.01 vs. the Model group. One-way ANOVA with Tukey's multiple comparisons test\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-3687044/v1/ac656800d955acaac759c47a.png"},{"id":48735162,"identity":"229736f3-b46d-4c32-921a-9e76ed07cdd6","added_by":"auto","created_at":"2023-12-24 02:22:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2408014,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3687044/v1/9d73b8fa-acde-4473-9f13-81036c88edda.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Discussion on Therapeutic Effect of ALA on Diabetic Peripheral Neuropathy rats from mitochondrial transport","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDiabetic peripheral neuropathy (DPN) is the most prevalent complication of diabetes mellitus (DM) and a major cause of disability. The most common form of DPN is distal symmetrical multiple peripheral neuropathy, which primarily affects sensory, motor, and autonomic nerves\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In severe cases, DPN can result in intense pain and even require amputation, significantly impacting the quality of life for patients\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Currently, there is no approved therapy or medication specifically for DPN.\u003c/p\u003e \u003cp\u003ePeripheral nerve demyelination and axonal degeneration are the primary pathological characteristics of DPN\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The survival of peripheral nerve axon ends heavily relies on energy support. Due to the neuron is a site of high energy demand and its unique polarized structure, it requires mitochondrial transport to maintain energy homeostasis throughout the cell, especially in the distal axon\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. To maintain axonal bioenergy, neurons deploy complex transport mechanisms that transport healthy mitochondria as a local energy source to the terminal and remove damaged mitochondria from the distal region\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Neurons maintain energy homeostasis in axons through mitochondrial transport. One of the motor proteins involved in the anterograde axonal mitochondrial transport is Kinesin family member 5A (KIF5A), which is crucial for maintaining the vitality and function of neurons\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Dynein is a crucial protein responsible for the reverse transport of mitochondria in axons of neurons. Its main function is to timely transport aging or damaged mitochondria at the end of axons back to the neuron cell body\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Dynein cytoplasmic 1 intermediate chain 2 (DYNC1I2) serves as the intermediate chain of Dynein.\u003c/p\u003e \u003cp\u003eThe transport of axon mitochondria is regulated in order to respond to changes in the biological energy status as neurons grow and become damaged, ensuring an adequate supply of ATP at these metabolically active locations. AMPK plays a crucial role in maintaining cell energy balance and has been found to be involved in various physiological activities\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Activation of AMPK can lead to the activation of CREB and enhance its transcription. CREB, as an important nuclear transcription factor, can upregulate KIF5A, thereby promoting forward mitochondrial transport \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.It has been observed that dysregulation of AMPK signaling and impaired axon energy metabolism are present in certain neurodegenerative diseases\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, there has been limited studies investigating the impact of AMPK on the sciatic nerve in the development of DPN.\u003c/p\u003e \u003cp\u003eDPN is a type of injury that affects nerves in a length-dependent manner, with early neuronal dysfunction potentially occurring in the distal axon. This suggests that the mechanism of mitochondrial transport plays a significant role in delivering mitochondria from the cell body to the axon for proper neuronal functioning.\u003c/p\u003e \u003cp\u003eALA, a derived antioxidant synthesized in mitochondria, serves as a cofactor\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. It exhibits a potent antioxidant effect and is frequently used in the prevention and treatment of DPN. ALA mediates the activation of AMPK by serine/threonine kinase (LKB1) or calmodulin-dependent protein kinase (CAMKK)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.Can ALA regulate mitochondrial axon transport and protect axons in diabetes by activating AMPK? The following experimental studies were carried out.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1.1 Animals\u003c/h2\u003e \u003cp\u003e The animal experiment was approved by the Guiding Prin-ciples for the Care and Use of Laboratory Animals published by the National Science and Technology Commission of China. Forty-five male Sprague-Dawley rats (7\u0026ndash;8 weeks old, 200\u0026ndash;240 g) were purchased from Liaoning Changsheng Biotechnology Co. LTD. (Beijing, China) (SCXK (Liaoning) 2020-0001) and housed in 15 cages in a facility with a 12-h/12-h artificial light\u0026ndash;dark cycle, a temperature of 22\u0026ndash;24\u0026deg;C, and a humidity level of 40%. The cage bedding was changed daily.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2 Induction of diabetes mellitus and drug treatment\u003c/h2\u003e \u003cp\u003eFifteen rats were randomly assigned to the control group and fed a standard diet. The remaining rats were fed a high-carbohydrate/high-fat diet for 6 weeks and were then intraperitoneally injected with STZ (Sigma-Aldrich, St Louis, MO, USA) (30 mg/kg). After 1 week, rats with a fasting glucose level\u0026thinsp;\u0026ge;\u0026thinsp;16.7 mmol/L were considered to be diabetic and continued to be fed a high-carbohydrate/high-fat diet. The diabetic rats were randomly divided into a DPN group and an ALA group, with 15 rats in each group. The rats in the ALA group were intragastrically administered an ALA suspension (60 mg/kg/day) for 12 weeks. The rats in the control group and DPN group were intragastrically administered an equal volume of distilled water every day for 12 weeks. After the treatment, the motor nerve conduction velocity and mechanical pain threshold of the diabetic rats in the ALA group and the DPN group were observed to be reduced to varying degrees: the rats in both the ALA group and the DPN group were therefore considered to have DPN\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.3 Blood glucose level and weight analysis\u003c/h2\u003e \u003cp\u003eThe diabetic rats having been identified, fasting glucose levels and body weight were measured and recorded every 4 weeks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.1.4 Mechanical pain threshold measurement\u003c/h2\u003e \u003cp\u003eThe mechanical pain threshold was measured by using an electronic von Frey (type 2390, 90 g probe, 0.8 mm diameter; IITC Life Science Inc. Woodland Hills, CA, USA) after the 12-week treatment period. The rats were placed on wire mesh, covered with a glass box, and allowed to acclimate to their surroundings for 15 min. A stainless-steel filament was applied vertically to the plantar surface of the hind paw. Rapid paw withdrawal by the rats was considered to be a positive response, and the force that elicited the response was recorded (grams). Paw withdrawal caused by the physical activity of the rats was considered to be a negative response. Measurements were taken at intervals of 3\u0026ndash;5 min. The response of each paw was assessed three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.1.5 Motor Nerve conduction velocity\u003c/h2\u003e \u003cp\u003eAfter the 12-week treatment period, the MNCV of the rats was measured. The rats were anesthetized with isoflurane (3% volume) before being fixed in the prone position. The sciatic nerve was exposed through an incision, and the skin between the biceps femoris and semitendinous muscles was separated from the muscles on the experimental side. Electrodes were implanted in two sites of the sciatic nerve notch approximately 10 mm apart. Platinum wire electrodes were placed directly under the sciatic nerve trunk in the right leg for stimulation, after which the data were recorded. The sciatic nerve was stimulated with a single square-wave pulse (intensity: 1.2 V, width: 1 ms) using an experimental system (BL-420s, Taimeng, Sichuan, China). The MNCV was measured and calculated as follows: MNCV\u0026thinsp;=\u0026thinsp;D/L (m/s).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.1.6 Immunofluorescence assay\u003c/h2\u003e \u003cp\u003eFor fluorescence staining, sciatic nerve and dorsal root ganglions sections underwent routine deparaffinization and rehydration and were then subjected to antigen retrieval by incubation with citric acid (0.01 mol/L, pH 6.0) in a microwave oven at approximately 90\u0026deg;C for 20 min. Next, the sections were blocked with non-immunoreactive serum and then incubated with antibodies against KIF5A (Abcam,USA,1:500), DYNC1I2 (Proteintech,USA,1:100), p-AMPK(Immunoway,USA,1:100), p-CREB(Abcam,USA,1:100) at 4\u0026deg;C overnight and subsequently incubated with secondary antibodies at 37\u0026deg;C for 1 h. After being stained, sections were incubated with DAPI for nuclear staining and sealed for photography using an inverted fluorescence microscope (CTS SP8, Leica, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.1.7 Western blotting for the detection of related proteins in tissues\u003c/h2\u003e \u003cp\u003eSciatic nerve and dorsal root ganglion tissues were homogenized in RIPA buffer, and protein samples were separated by 10% SDS\u0026ndash;PAGE and transferred onto nitrocellulose membranes (Pall Gelman, Ann Arbor, MI, USA). After transfer, the membranes were blocked with 5% skim milk powder. Then, the membranes were incubated with antibodies against KIF5A(Abcam,USA,1:1000), DYNC1I2(Proteintech,USA,1:500), AMPK(Immunoway,USA,1:1000), p-AMPK(Immunoway,USA,1:1000), CREB(Proteintech,USA,1:500), p-CREB(Abcam,USA,1:500) at 4\u0026deg;C overnight followed by a fluorescent secondary antibody for 1 h at room temperature. A laser infrared scanner (Odyssey, LI-COR, USA) was used to visualize the protein bands.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Cell culture assays in vitro\u003c/h2\u003e \u003cp\u003eNSC34 cells were maintained in culture media with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin and streptomycin at 37\u0026deg;C in an incubator with a humidified atmosphere and 5% CO2. NSC34 cells were divided into the Control group, DPN group, ALA group. Except the Control group, the other groups were added 50mmol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose and 250\u0026micro;mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e palmitic acid. ALA group were given 250\u0026micro;M ALA\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Relevant indexes were detected 24h after administration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Immunofluorescence analysis\u003c/h2\u003e \u003cp\u003eThe dried and sterilized glass slides were placed on a 24-well petri dish, the cells were spread evenly on the glass slides in the wells, and 4% paraformaldehyde was added and incubated for 20 min at room temperature. Then 0.25% Triton X-100 was added and incubated for 15 min for permeabilization, and 10% normal goat serum was added and incubated for 30 min for blocking. Subsequently, the cells were incubated with the primary antibody KIF5A(Abcam,USA,1:1000), DYNC1I2(Proteintech,USA,1:500), AMPK(Immunoway,USA,1:1000), p-AMPK(Immunoway,USA,1:1000), CREB(Proteintech,USA,1:500), p-CREB(Abcam,USA,1:500) overnight at 4\u0026deg;C, followed by the relevant secondary antibody at 37\u0026deg;C in the dark for 1 h for fluorescence staining. DAPI was used for nuclear staining. β-tubulin Ⅲ was used to measure and characterize the length of neurons and axons. After immunofluorescence staining, 15 different neurons were randomly selected from each group of cells, and the axonal length of the neurons was detected using Image J.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Western blotting for the detection of related proteins in cells\u003c/h2\u003e \u003cp\u003eNSC34C cells were homogenized in RIPA buffer, and protein samples were separated by 10% SDS\u0026ndash;PAGE and transferred onto nitrocellulose membranes (Pall Gelman, Ann Arbor, MI, USA). After transfer, the membranes were blocked with 5% skim milk powder. Then, the membranes were incubated with antibodies against KIF5A(Abcam,USA,1:1000), DYNC1I2(Proteintech,USA,1:500), AMPK(Immunoway,USA,1:1000), p-AMPK(Immunoway,USA,1:1000), CREB(Proteintech,USA,1:500), p-CREB(Abcam,USA,1:500) at 4\u0026deg;C overnight followed by a fluorescent secondary antibody for 1 h at room temperature. A laser infrared scanner (Odyssey, LI-COR, USA) was used to visualize the protein bands.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Statistical analysis\u003c/h2\u003e \u003cp\u003eSPSS 24.0 (IBM Corp. Released 2016. IBM SPSS Statistics for Windows, Version 24.0. Armonk, NY: IBM Corp) was used to analyze the data. All data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (M\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). Normally distributed data were analyzed with one-way ANOVA with Tukey\u0026rsquo;s test for multigroup independent samples. Statistical tests were performed using two-sided tests. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1 Body weight and fasting blood glucose levels\u003c/h2\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eEffects of ALA on body weight in rats (g)\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGroups\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0 Weeks\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e4 Weeks\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e8 Weeks\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e12 Weeks\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eControl\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e330.38\u0026thinsp;\u0026plusmn;\u0026thinsp;9.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e389.91\u0026thinsp;\u0026plusmn;\u0026thinsp;13.30\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e405.83\u0026thinsp;\u0026plusmn;\u0026thinsp;14.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e412.54\u0026thinsp;\u0026plusmn;\u0026thinsp;13.98\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDPN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e356.36\u0026thinsp;\u0026plusmn;\u0026thinsp;18.95\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e415.29\u0026thinsp;\u0026plusmn;\u0026thinsp;10.94\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e416.29\u0026thinsp;\u0026plusmn;\u0026thinsp;17.63\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e414.54\u0026thinsp;\u0026plusmn;\u0026thinsp;14.03\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eALA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e332.87\u0026thinsp;\u0026plusmn;\u0026thinsp;13.26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e407.48\u0026thinsp;\u0026plusmn;\u0026thinsp;16.32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e413.49\u0026thinsp;\u0026plusmn;\u0026thinsp;8.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e420.86\u0026thinsp;\u0026plusmn;\u0026thinsp;15.61\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003eValues are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, n\u0026thinsp;=\u0026thinsp;15 for each group\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eDPN diabetic peripheral neuropathy, ALA \u0026alpha;-lipoic acid.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eEffects of ALA on fasting blood glucose in rats (mmol/L)\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGroups\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e0 Weeks\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e4 Weeks\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e8 Weeks\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e12 Weeks\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eControl\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e4.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e4.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e4.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e4.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDPN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e26.26\u0026thinsp;\u0026plusmn;\u0026thinsp;2.53\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e28.56\u0026thinsp;\u0026plusmn;\u0026thinsp;2.49\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e28.92\u0026thinsp;\u0026plusmn;\u0026thinsp;2.54\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e26.70\u0026thinsp;\u0026plusmn;\u0026thinsp;4.55\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eALA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e27.43\u0026thinsp;\u0026plusmn;\u0026thinsp;3.93\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e28.27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.34\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e26.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e26.71\u0026thinsp;\u0026plusmn;\u0026thinsp;2.56\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003eValues are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, n\u0026thinsp;=\u0026thinsp;15 for each group\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e# # P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. the control group. One-way ANOVA with Tukey\u0026rsquo;s multiple comparisons test.\u003c/p\u003e\n\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, the body weight of the rats that were fed a high-carbohydrate/high-fat diet was significantly increased after 4 weeks. As shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, fasting blood glucose levels were significantly higher in both DPN and ALA group compared with Control group. Compared with DPN group, there was no significant difference in fasting blood glucose in ALA group.\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that reducing blood glucose levels does not slow down the progression of the disease Although ALA has a mild hypoglycemic effect, our experiment found no significant impact of ALA on reducing blood glucose levels in rats. Therefore, the therapeutic effect of ALA on DPN was not achieved through the reduction of blood glucose levels.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2 Pain thresholds\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eEffects of ALA on MNCV, the paw withdrawal threshold in rats\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eGroups\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePTW(g)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMNCV(m/s)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eControl\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e79.84\u0026thinsp;\u0026plusmn;\u0026thinsp;5.62\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e53.53\u0026thinsp;\u0026plusmn;\u0026thinsp;3.28\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDPN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e23.92\u0026thinsp;\u0026plusmn;\u0026thinsp;6.26\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e29.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.35\u003csup\u003e##\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eALA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e50.93\u0026thinsp;\u0026plusmn;\u0026thinsp;7.85\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\"\u0026plusmn;\"\u003e\n\u003cp\u003e38.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"3\"\u003eValues are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, n\u0026thinsp;=\u0026thinsp;15 for each group\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"3\"\u003eMNCV motor nerve conduction velocity\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e## P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. the control group; ** P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 vs. the DPN group. One-way ANOVA with Tukey\u0026rsquo;s multiple comparisons test.\u003c/p\u003e\n\u003cp\u003eAs illustrated in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, the rats in the DPN group were more sensitive to mechanical stimulation than the normal rats (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01); however, the mechanical thresholds for paw withdrawal of the ALA-treated rats were markedly increased compared with those of the rats in the DPN group(P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n\u003ch2\u003e3.3 MNCV\u003c/h2\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003eAs shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, the MNCV of the DPN group was clearly lower than those of the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the MNCV of the ALA group were significantly higher than those of the DPN group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/div\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4 Pathological changes of sciatic nerves(HE)\u003c/h2\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, HE staining microscopy showed that the structure of nerve fibers in the sciatic nerve in the Control group was normal, without swelling or atrophy of axons, and the structure of myelin sheath was complete. In DPN group, there was a large amount of swelling of nerve axons, partial atrophy, and large loss of myelin sheath. The axonal swelling and demyelination in ALA group was less, and axonal atrophy was not obvious.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\n\u003ch2\u003e3.5 Immunofluorescence and western blot results in vivo\u003c/h2\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, b, c, the expression of KIF5A in the DPN group was decreased compared with that in the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Additionally, KIF5A levels were increased in the ALA group compared with the DPN group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results indicate that ALA can increase the level of anterograde mitochondrial transport in rats with DPN. The trends revealed by western blotting were consistent with the expression data obtained by immunofluorescence.\u003c/p\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;3a, b, c, the expression of DYNC1I2 in the DPN group was increased compared with that in the control group(P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Additionally, DYNC1I2 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) levels were decreased in the ALA group compared with the DPN group. These results indicate that ALA can decrease the level of retrograde mitochondrial transport in rats with DPN. The trends revealed by western blotting were consistent with the expression data obtained by immunofluorescence.\u003c/p\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea, b, c,the expression of p-AMPK/AMPK in the DPN group was decreased compared with that in the control group(P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Additionally, the p-AMPK/AMPK levels were increased in the ALA group compared with the DPN group(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results indicate that ALA can increase the level of p-AMPK in rats with DPN. The trends revealed by western blotting were consistent with the expression data obtained by immunofluorescence.\u003c/p\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, b, c, the expression of p-CREB/CREB in the DPN group was decreased compared with that in the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, the p-CREB/CREB levels were increased in the ALA group compared with the DPN group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results indicate that ALA can increase the level of p-CREB, which is a downstream factor of p-AMPK, in rats with DPN. The trends revealed by western blotting were consistent with the expression data obtained by immunofluorescence.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\n\u003ch2\u003e3.6 Immunofluorescence and western blot results in vitro\u003c/h2\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea, b, the neuron axon length in the Model group was decreased compared with that in the control group(P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Additionally, neuron axon length levels were increased in the ALA group compared with the Model group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results indicate that ALA can increase the axon length in Model group of NSC34 cells.\u003c/p\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;7a, b, c,the expression of KIF5A in the Model group was decreased compared with that in the control group(P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Additionally, KIF5A levels were increased in the ALA group compared with the Model group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results indicate that ALA can increase the level of anterograde mitochondrial transport in Model group of NSC34 cells. The trends revealed by western blotting were consistent with the expression data obtained by immunofluorescence.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ea, b, c, the expression of DYNC1I2 in the Model group was increased compared with that in the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Additionally, DYNC1I2 levels were decreased in the ALA group compared with the Model group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results indicate that ALA can decrease the level of retrograde mitochondrial transport in Model group of NSC34 cells. The trends revealed by western blotting were consistent with the expression data obtained by immunofluorescence.\u003c/p\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003ea, b, c, the expression of p-AMPK/AMPK in the Model group was decreased compared with that in the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Additionally, the p-AMPK levels were increased in the ALA group compared with the Model group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results indicate that ALA can increase the level of p-AMPK in Model group of NSC34 cells. The trends revealed by western blotting were consistent with the expression data obtained by immunofluorescence.\u003c/p\u003e\n\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003ea, b, c, the expression of p-CREB in the Model group was decreased compared with that in the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Additionally, the p-CREB levels were increased in the ALA group compared with the Model group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results indicate that ALA can increase the level of p-CREB, which is a downstream factor of p-AMPK, in Model group of NSC34 cells. The trends revealed by western blotting were consistent with the expression data obtained by immunofluorescence.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe mechanism of DPN is not fully understood and treatments are still being explored. ALA is a potent antioxidant that can effectively eliminate various reactive oxygen species. This effect has been applied to the prevention and treatment of DPN\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. But we overlooked another aspect of ALA's efficacy: AMPK agonists. The purpose of this study was to investigate the protective effect of ALA on peripheral nerves in DPN rats based on AMPK activation. Our results suggest that ALA can regulate the CREB/KIF5A pathway by activating AMPK, improve mitochondrial forward transport, protect sciatic nerve axon terminus, and prevent DPN.\u003c/p\u003e \u003cp\u003eAMPK is a cellular energy sensor expressed by a variety of organs and is closely related to metabolic diseases such as cancer, obesity and diabetes\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The role of AMPK pathway in the prevention and treatment of diabetic complications has received increasing attention in recent years. CREB is the downstream target of AMPK, and as a crucial nuclear transcription factor, it can increase the level of kinase protein subunits when activated\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Numerous studies have demonstrated a synergistic relationship between AMPK and CREB and their involvement in adaptive responses to cellular energy stress. In the AMPK/CREB metabolic control network, p-AMPK can activate CREB, thereby regulating the expression of the anterograde axon mitochondrial transport protein KIF5A\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur results show that in DPN rats, AMPK activation is decreased, CREB activation is down-regulated, and the expression of mitochondrial anterograde transport protein KIF5A is reduced, resulting in insufficient axon energy supply and neurological dysfunction. The expression of mitochondrial retrograde transport protein DYNC1I2 increased in DPN rats, which may be related to the increase of mitochondrial damage caused by high sugar. At present, the relationship between AMPK activation and DYNC1I2 expression has not been reported, which can be further explored in future studies.\u003c/p\u003e \u003cp\u003eIn this study, p-AMPK/AMPK ratio and p-CREB/CREB and KIF5A levels were up-regulated in ALA group. This suggests that ALA activates AMPK, activates its downstream target CREB, enhances the expression of KIF5A, improves the energy supply of terminal axons, and alleviates peripheral nerve injury in DPN rats. The downregulation of DYNC1I2 expression may be related to the reduction of mitochondrial damage, which is caused by the increased energy supply of terminal axons and the antioxidant effect of ALA. The peripheral neuroprotective effect of ALA is related to its regulation of mitochondrial transport at the end of axons.\u003c/p\u003e \u003cp\u003eIn vitro experiments, NSC34 cells were cultured in an environment high in sugar and fat. The NSC34 cell line is characterized by longer axons and is able to respond more significantly to axon-related damage and changes in mitochondrial transport. The results of in vitro experiments are consistent with those of animal experiments, suggesting that ALA may regulate the anteroonal transport of axon mitochondria through AMPK/CREB pathway, enhance the energy supply at the end of axon, and ultimately play a neuroprotective role.\u003c/p\u003e \u003cp\u003eHowever, it is important to interpret the experimental results with caution when extrapolating treatment for diabetic peripheral neuropathy in humans because of the inevitable physiological and anatomical differences between rats and humans.\u003c/p\u003e \u003cp\u003eIn summary, our study suggests that ALA regulates mitochondrial transport by activating AMPK, effectively alleviating DPN-related symptoms. These findings provide preliminary evidence for the pharmacological effect of ALA on DPN.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe findings of this study suggest that ALA has the potential to protect against DPN by regulating mitochondrial transport and improving peripheral nerve function in rats. These effects are thought to be mediated by AMPK. It is possible that ALA may also have a similar mechanism of action in the human sciatic nerve.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJiaxin Tian, Jingwen An Literature search and data collection, investigation, data collation, analysis, and writing-first draft, compilation of all figures. Linchun Song, WangZhang, DieChen, Tianya Zhang Search and data collection, analysis, and writing-first draft. Ying Ben review and editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation [grant numbers 81803922] funded by CHINA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e This project was approved by the Ethics Committee of Hebei University of Traditional Chinese Medicine (DWLL2018042).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e Informed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZherebitskaya E, Akude E, Smith DR, Fernyhough P (2009) Development of selective axonopathy in adult sensory neurons isolated from diabetic rats: role of glucose-induced oxidative stress. Diabetes 58(6):1356\u0026ndash;1364\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu Y (2021) Gold Standard for Diagnosis of DPN. Front Endocrinol 12:719356\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiyashita A, Kobayashi M, Yokota T, Zochodne DW (2023) Diabetic Polyneuropathy: New Strategies to Target Sensory Neurons in Dorsal Root Ganglia. Int J Mol Sci \u003cem\u003e24\u003c/em\u003e (6)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSheng ZH (2017) The Interplay of Axonal Energy Homeostasis and Mitochondrial Trafficking and Anchoring. Trends Cell Biol 27(6):403\u0026ndash;416\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSheng ZH (2014) Mitochondrial trafficking and anchoring in neurons: New insight and implications. J Cell Biol 204(7):1087\u0026ndash;1098\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng XT, Sheng ZH (2021) Developmental regulation of microtubule-based trafficking and anchoring of axonal mitochondria in health and diseases. Dev Neurobiol 81(3):284\u0026ndash;299\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKonjikusic MJ, Gray RS, Wallingford JB (2021) The developmental biology of kinesins. Dev Biol 469:26\u0026ndash;36\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCanty JT, Tan R, Kusakci E, Fernandes J, Yildiz A (2021) Structure and Mechanics of Dynein Motors. Annual Rev Biophys 50:549\u0026ndash;574\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerzig S, Shaw RJ (2018) AMPK: guardian of metabolism and mitochondrial homeostasis. Nat Rev Mol Cell Biol 19(2):121\u0026ndash;135\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTao K, Matsuki N, Koyama R (2014) AMP-activated protein kinase mediates activity-dependent axon branching by recruiting mitochondria to axon. Dev Neurobiol 74(6):557\u0026ndash;573\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVingtdeux V, Davies P, Dickson DW, Marambaud P (2011) AMPK is abnormally activated in tangle- and pre-tangle-bearing neurons in Alzheimer's disease and other tauopathies. Acta Neuropathol 121(3):337\u0026ndash;349\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePathak D, Berthet A, Nakamura K (2013) Energy failure: does it contribute to neurodegeneration? Ann Neurol 74(4):506\u0026ndash;516\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePacker L, Cadenas E (2011) Lipoic acid: energy metabolism and redox regulation of transcription and cell signaling. J Clin Biochem Nutr 48(1):26\u0026ndash;32\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTargonsky ED, Dai F, Koshkin V, Karaman GT, Gyulkhandanyan AV, Zhang Y, Chan CB, Wheeler MB (2006) alpha-lipoic acid regulates AMP-activated protein kinase and inhibits insulin secretion from beta cells. Diabetologia 49(7):1587\u0026ndash;1598\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBiessels GJ, Bril V, Calcutt NA, Cameron NE, Cotter MA, Dobrowsky R, Feldman EL, Fernyhough P, Jakobsen J, Malik RA, Mizisin AP, Oates PJ, Obrosova IG, Pop-Busui R, Russell JW, Sima AA, Stevens MJ, Schmidt RE, Tesfaye S, Veves A, Vinik AI, Wright DE, Yagihashi S, Yorek MA, Ziegler D, Zochodne DW (2014) Phenotyping animal models of diabetic neuropathy: a consensus statement of the diabetic neuropathy study group of the EASD (Neurodiab). J peripheral Nerv system: JPNS 19(2):77\u0026ndash;87\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuo YT, Lin TH, Chen WL, Lee HM (2012) Alpha-lipoic acid induces adipose triglyceride lipase expression and decreases intracellular lipid accumulation in HepG2 cells. Eur J Pharmacol \u003cem\u003e692\u003c/em\u003e (1\u0026ndash;3), 10\u0026thinsp;\u0026ndash;\u0026thinsp;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBondar A, Popa AR, Papanas N, Popoviciu M, Vesa CM, Sabau M, Daina C, Stoica RA, Katsiki N, Stoian AP (2021) Diabetic neuropathy: A narrative review of risk factors, classification, screening and current pathogenic treatment options (Review). Experimental and therapeutic medicine 22(1):690\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuiroz-Aldave J, Durand-V\u0026aacute;squez M, Gamarra-Osorio E, Suarez-Rojas J, Jantine Roseboom P, Alcal\u0026aacute;-Mendoza R, Coronado-Arroyo J, Zavaleta-Guti\u0026eacute;rrez F, Concepci\u0026oacute;n-Urteaga L, Concepci\u0026oacute;n-Zavaleta M (2023) Diabetic neuropathy: Past, present, and future. Caspian J Intern Med 14(2):153\u0026ndash;169\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEvans JL, Goldfine ID (2000) Alpha-lipoic acid: a multifunctional antioxidant that improves insulin sensitivity in patients with type 2 diabetes. Diabetes Technol Ther 2(3):401\u0026ndash;413\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKamenova P (2006) Improvement of insulin sensitivity in patients with type 2 diabetes mellitus after oral administration of alpha-lipoic acid. Hormones (Athens Greece) 5(4):251\u0026ndash;258\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCapece U, Moffa S, Improta I, Di Giuseppe G, Nista EC, Cefalo CMA, Cinti F, Pontecorvi A, Gasbarrini A, Giaccari A, Mezza T (2022) Alpha-Lipoic Acid and Glucose Metabolism: A Comprehensive Update on Biochemical and Therapeutic Features. Nutrients \u003cem\u003e15\u003c/em\u003e (1)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePapanas N, Ziegler D (2014) Efficacy of α-lipoic acid in diabetic neuropathy. Expert Opin Pharmacother 15(18):2721\u0026ndash;2731\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSteinberg GR, Hardie DG (2023) New insights into activation and function of the AMPK. Nat Rev Mol Cell Biol 24(4):255\u0026ndash;272\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePuthanveettil SV, Monje FJ, Miniaci MC, Choi YB, Karl KA, Khandros E, Gawinowicz MA, Sheetz MP, Kandel ER (2008) A new component in synaptic plasticity: upregulation of kinesin in the neurons of the gill-withdrawal reflex. Cell 135(5):960\u0026ndash;973\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alpha-lipoic acid, Diabetic peripheral neuropathy, Sciatic nerve AMPK","lastPublishedDoi":"10.21203/rs.3.rs-3687044/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3687044/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eTo investigate the AMPK/CREB pathway\u0026ndash;mediated effect of alpha-lipoic acid (ALA) on the sciatic nerve of rats with diabetic peripheral neuropathy (DPN) and to attempt to elucidate the underlying mechanism.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn vivo experiment, healthy male Sprague-Dawley (SD) rats were induced by high-carbohydrate/high-fat diet and intraperitoneal injection of streptozotocin (STZ) (30 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) to induce diabetes. The diabetes SD rats were randomly divided into DPN group and alpha lipoic acid (ALA) group (n\u0026thinsp;=\u0026thinsp;15). The other 15 SD rats were set as Control group. Then Control group and DPN group received the same amount of normal saline by intragastric administration, and ALA groups received ALA intervention every day for 12 weeks. Motor nerve conduction velocity (MNCV) and Paw Withdrawal Threshold (PWT) were detected. The morphological changes were observed by HE staining in sciatic nerves. Kinesin family member 5A (KIF5A), Dynein Cytoplasmic 1 Intermediate Chain 2 (DYNC1I2), phosphorylated Adenosine 5'-monophosphate (AMP) activated protein kinase (p-AMPK), Adenosine 5'-monophosphate (AMP) activated protein kinase (AMPK), phosphorylated cAMP responsive element binding protein(p-CREB) and cAMP responsive element binding protein(CREB)were observed by immunofluorescence assay and Western blot. In the vitro cell experiment, the NSC34 cells injury model was established by treating with 50 mmol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of high glucose and 250 \u0026micro;mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of palmitic acid sodium. NSC34 cells were randomly divided into Control group, Model group and alpha lipoic acid intervention group (ALA group). ALA group was given corresponding ALA for 24 hours of intervention. The axon changes were observed by measured neuron axon length in NSC34 cells. KIF5A, DYNC1I2, p-AMPK,AMPK, p-CREB and CREB were observed by immunofluorescence assay and Western blot.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eALA improved the MNCV and PTW of rats with DPN and reduced their mechanical pain threshold. AMPK was activated by ALA. p-CREB, KIF5A expression was upregulated, while DYNC1I2 expression was downregulated. ALA regulates mitochondrial transport in peripheral nerve.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eALA activates CREB and KIF5A through AMPK, regulates positive mitochondrial transport, protects axons, and attenuates DPN.\u003c/p\u003e","manuscriptTitle":"Discussion on Therapeutic Effect of ALA on Diabetic Peripheral Neuropathy rats from mitochondrial transport","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-12-04 21:26:48","doi":"10.21203/rs.3.rs-3687044/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5ef16371-0460-4801-a5be-55624055e687","owner":[],"postedDate":"December 4th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-12-24T02:14:10+00:00","versionOfRecord":[],"versionCreatedAt":"2023-12-04 21:26:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3687044","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3687044","identity":"rs-3687044","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}