According to the latest International Diabetes Federation statistics for 2021, the total number of people with diabetes worldwide is 536.6 million—and China accounts for a quarter (1). The prevalence of diabetes among adults in China is 12.8%, higher than the global average of 10.5%. Moreover, the number of people with diabetes in China is approximately 129.8 million, and those with type 2 diabetes mellitus (T2DM) account for > 90%. Therefore, T2DM is a serious public health problem in China (2). Additionally, the chronic complications caused by T2DM are a threat to national health. Notably, the complications involving peripheral nerves—peripheral neuropathy—can affect 75% of people with T2DM. Peripheral neuropathy occurs as early as in the pre-diabetic state of obesity, indicating its close relation to diabetes, obesity, and other metabolic diseases (3, 4).

Distal symmetrical polyneuropathy (DSPN) is the most representative manifestation of nerve damage in T2DM and is more common than other spinal nerves, cranial nerves, and autonomic neuropathy lesions (5). DSPN develops slowly, starting with small nerve fibers at the end of the limb and progressing to larger nerves, including sensory and motor nerves. The symmetrical onset of numbness and sensory abnormalities is the earliest clinical symptom in many patients with DSPNs. Moreover, approximately one-third of patients experience burning, pin and needle sensations, and pain. These painful symptoms are unbearable and lead to insomnia and depression, reducing patients’ quality of life (6, 7). Additionally, patients with DSPN are prone to complications, such as diabetic foot ulcers, due to a long-term lack of self-sensory protection, leading to serious complications, such as irreversible amputation and shortened life expectancy. Finally, as the disease progresses, people’s ability to work becomes severely affected, causing a serious economic burden and wastage of resources for individuals, families, and society; therefore, DSPN requires more attention (8, 9).

The clinical efficacy of DSPN treatment is poor. The Action to Control Cardiovascular Risk in Diabetes (ACCORD) study showed that glycemic control alone in T2DM does not reduce the incidence of DSPN, suggesting that hyperglycemia is not the only factor influencing the development of DSPN. Recently, clinicians have been looking for alternative treatments to improve DSPN (10). The etiology and pathogenesis of DSPN have not been elucidated; however, its development involves or is accelerated by abnormal insulin signaling pathways, microcirculatory disorders, non-enzymatic advanced glycosylation end product pathways, oxidative stress, and other factors leading to mitochondrial dysfunction, DNA damage, and apoptosis (9, 11).

Vitamin D is a fat-soluble ring-opening steroid discovered over 100 years ago and includes vitamin D₂ (ergocalciferol) and D₃ (cholecalciferol). Vitamin D₂ is derived from foods, such as mushrooms, offal, egg yolks, and fatty sea fish. Vitamin D₃ is converted from 7-dehydrocholesterol in the skin by ultraviolet light. Vitamins D₂ or D₃ require two hydroxylation processes, mediated by 25-hydroxylase in the liver and 1α-hydroxylase in the kidneys, to become active as 1,25-(OH)₂D₂ or 1,25-(OH)₂D₃, respectively. At present, activated vitamin D has gone beyond the traditional concept of vitamin and as it can play a hormone-like role, so 1,25-(OH)₂D is also known as D hormone. Therefore, some scholars refer to the unactuated form of vitamin D as a D prohormone. Additionally, activated vitamin D is directly involved in gene regulation via the vitamin D receptor (VDR), which has several biological roles. Since vitamin D receptors are found in bones, kidneys, parathyroid glands, small intestine, nerve tissue, and pancreatic β-cell, vitamin D may have other biological roles in addition to its traditional role of regulating calcium, phosphorus, and bone metabolism. These roles include facilitating cell differentiation and proliferation, reducing superoxide, regulating lipid metabolism and inflammatory factors, reducing superoxide, promoting nerve growth factors, regulating immunity, reducing cytokine storms, and reducing COVID-2019 mortality (12–16).

Considering all these functions, vitamin D is important for health; however, its insufficiency is a widespread public health problem. Vitamin D nutritional status is determined by measuring serum 25-(OH)D levels—a sum of 25-(OH)D₂ and 25-(OH)D₃. The Endocrine Society recommends a 25-(OH)D level of 30 ng/mL (17); a patient is considered to have a vitamin D -insufficient state if this level is not reached (17–19). Studies have shown that vitamin D deficiency occurs in 74.7% of people with T2DM and affects people with prediabetes. Moreover, studies have shown that people with low vitamin D levels and prediabetes have a significantly higher risk of developing diabetes (20, 21).

A study by the National Health and Nutrition Examination Survey showed that the risk of developing DSPN increases by 2.59 times in people with vitamin D deficiency (22). Another study suggested that vitamin D deficiency may be an independent risk factor for DSPN (23). In an observational study, Seham et al. (24) found that vitamin D levels were significantly lower in patients and DSPN than in those with T2DM without DSPN and confirmed that 87.6% of those with DSPN had vitamin D deficiency, and the prevalence of vitamin D deficiency in T2DM without DSPN was only 45%.

However, the mechanisms by which vitamin D deficiency specifically affects DSPN are unknown. Vitamin D may improve glycemic control by promoting β-cell secretion and increasing insulin sensitivity. Additionally, vitamin D may reduce oxidative stress and prevent nerve damage (25). Animal studies have shown that vitamin D increases the synthesis of nerve growth factors in rats (26). Vitamin D is a potent inducer of neurotransmitters and can reduce nerve demyelination and improve axonal regeneration (27, 28).

A meta-analysis showed that vitamin D supplementation effectively reduced neuropathic pain and prevented further nerve damage (29). The association between vitamin D and pain may be due to nociceptive calcitonin gene-related peptide (CGRP)-positive neurons. These neurons have a distinct vitamin D phenotype, and their ligands and receptors are hormonally regulated. A study reported that vitamin D receptor expression in the growth cones and CGRP expression increased rapidly with vitamin D deficiency, indicating a possible connection between vitamin D and pain. Additionally, this study suggested the presence of lower pain tolerance in patients with vitamin D deficiency (30, 31).

Adequate vitamin D levels are not achieved with sunlight and food intake alone; therefore, additional supplements are required. Studies have shown that different doses of vitamin D supplementation may produce different results in DSPN treatment. A clinical study by Karonova et al. (32) showed that high-dose vitamin D (HDVD) supplementation in patients with DSPN and vitamin D deficiency achieved adequate status in all patients; however, only half of the low-dose group achieved adequate status. Moreover, the severity of neuropathy was accompanied by a significant reduction in inflammatory markers in the HDVD group; however, these markers were not altered in the low-dose group. Basit et al. (33) showed a significant decrease in total pain score and a reduction in positive symptoms in diabetic neuropathy patients treated with HDVD. Silva et al. (34) treated patients who had diabetic cardiac autonomic neuropathy with HDVD and showed significant improvement in parameters related to resting heart rate variability without adverse effects. Notably, neuropathy repair and symptom improvement are slow; therefore, we decided to use HDVD supplementation to repair neuropathy.

It is currently believed that vitamin D₂ and D₃ supplementation have the same effect of correcting vitamin D insufficiency, and it is believed that injection is better than oral supplementation (17, 35). Indeed, lower daily doses of vitamin D supplements require longer time to increase vitamin D levels in the body and are less likely to sustain sufficient levels. In addition, daily medication reduced compliance and executive function in patients, thereby reducing the number of people who reach sufficient state of vitamin D. Therefore, some scholars have proposed that the injection of HDVD supplementation as a method of administration with lower frequency and longer interval will be more conducive to obtain desired intervention outcomes (36, 37). Compared to vitamin D₃, vitamin D₂ has a lower binding affinity to vitamin D-binding proteins (VDBP). This means that taking vitamin D₂ instead of vitamin D₃ will result in higher levels of free vitamin D concentration in the blood, which will bind better to VDR (38, 39). Indeed, vitamin D₂ has been used clinically for more than 50 years (39). Therefore, we use vitamin D₂ injection as a strategy to correct vitamin D insufficiency.

The traditional nerve conduction test reflects only the lesions of large nerve fibers but not those of small fibers, such as fine myelinated A δ, unmyelinated C, and autonomic nerve fibers, which mediate nociceptive pain and temperature perception (5). Michigan Neuropathy Screening Instrument (MNSI) comprises a questionnaire and physical examination. The questionnaire contains 15 questions that address DSPN symptoms, with a maximum score of 13 points, and is completed by the patients. The physical examination is performed by the physician and includes ankle reflexes, vibration perception, foot ulceration, and feet appearance. Physical examination has a maximum score of 8 points for both feet. Multiple studies have demonstrated that MNSI is a reliable and effective tool for assessing DSPN. Additionally, compared with nerve conduction tests, MNSI avoids invasive examinations and the discomfort of electrical stimulation. Moreover, MNSI may avoid missing early small-fiber lesions in DSPN (40–44). Therefore, we will use the MNSI to assess the changes in neuropathy after HDVD supplementation.

The development of DSPN is relatively reversible in the early stage; however, it becomes a refractory nerve injury in the later stage, and the condition is persistent and recurrent. Correcting vitamin D deficiency helps prevent and treat DSPN. However, the specific dosage, long-term treatment effects, and related side effects should be confirmed in high-quality randomized controlled trials. Few clinical trials have investigated the effects of vitamin D supplementation on neuropathy in China’s population with DSPN and T2DM. Therefore, this study aims to explore the efficacy of HDVD supplementation in treating DSPN in T2DM, assess its safety, and collect information on adverse reactions.

DSPN can induce diabetic foot ulcer and amputation, increasing the social burden and consuming medical resources. The United States spends > $10 billion yearly to treat DSPN. Early identification and treatment of DSPN can save at least 80% of medical costs (50–52). Therefore, preventing the further development of DSPN has attracted our attention.

Vitamin D deficiency is a growing global health problem, even in countries like the United Arab Emirates, which receives plenty of sunshine yearly (18). This implies that the vitamin D produced by the sun’s ultraviolet rays is insufficient. A recent meta-analysis showed that vitamin D deficiency in T2DM increased the risk of DSPN by 1.22 times (53). This evidence highlights an early treatment window for patients with T2DM and the possibility that vitamin D supplementation may prevent the development of DSPN. A previous retrospective study found that vitamin D levels in patients with T2DM and DSPN were one-third lower than those without DSPN, suggesting that vitamin D may affect DSPN progression (18). Furthermore, clinical studies have shown that HDVD supplementation can significantly reduce the pain score of DSPN, reduce demyelination, improve axonal regeneration, and even improve the quality of life of patients with DSPN (33, 34, 54).

Notably, patients with vitamin D deficiency may not rely solely on increased sun exposure and dietary supplements; therefore, additional vitamin D supplements are needed. Supplementation is achieved mainly by oral administration and intramuscular injection of vitamin D pharmaceutical preparations. However, recovery from neuropathy is slow, and most patients have difficulty maintaining a daily low dose of vitamin D supplements. Moreover, it also takes longer to achieve vitamin D sufficiency with low-dose supplementation.

Since low-dose vitamin D supplementation cannot quickly correct this deficiency, some scientists have begun to try HDVD supplementation programs. A clinical study conducted by Masood et al. (55) with a single dose of 600000 units of vitamin D showed that it took only 2 months for 70% of people with vitamin D deficiency to achieve an adequate status, indicating that high-dose vitamin D supplementation can correct vitamin D deficiency quickly. This means the high-dose regimen could reduce the time traditionally required to reach the target. Additionally, a previous study by Xu et al. (56) demonstrated that a single injection of 600000 units of vitamin D increased 25(OH)D level by 10.3 ng/mL over 12 weeks, indicating that HDVD injection therapy is effective and durable in raising vitamin D concentrations. A clinical study by Diamond et al. (35) showed that a regimen of 600000 units of vitamin D injected intramuscularly once yearly increased participants’ vitamin D levels by an average of 128%, and all participants’ vitamin D levels were normalized. Furthermore, a bioavailability study by Cipriani et al. (57) revealed that vitamin D levels peaked on day 120 after a single intramuscular dose of 600000 units of vitamin D, and the conversion to 1,25 (OH)₂D was more stable and less volatile after intramuscular administration than after oral administration. Overall, the studies described above suggest that intramuscular HDVD is a more effective method of vitamin D supplementation than daily oral low-dose vitamin D supplementation. Moreover, the HDVD regimen has better compliance owing to fewer supplements, no need to take medication daily, and less forgetting or omission. However, few studies have investigated whether an HDVD can improve peripheral neuropathy. Our study aims to determine the efficacy of HDVD in T2DM patients with DSPN.

To make our study feasible, our study plan will exclude patients with severe renal insufficiency. Because 1α-hydroxylase is tightly regulated in the body, safe dose range for vitamin D supplementation is wide and there is no excess of 1,25-(OH)₂D that is formed in the body. Previous studies have shown that poisoning is only possible when the 25-hydroxyvitamin D levels is greater than 224 ng/mL (560nmol/L) (17, 58, 59). A prospective randomized controlled study by Mehta et al. showed significant improvement in neuropathy in patients with DSPN who received HDVD supplementation for 6 months. The study included patients with chronic kidney disease (CKD) stages 1-4 and excluded patients with CKD stage 5 (eGFR <15 mL/min/1.73 m²). The authors showed that 25-hydroxyvitamin D levels were significantly increased 6 months after the HDVD supplementation (before: 18.91 ± 5.3 ng/mL vs. after: 53.71 ± 7.8 ng/mL, p<0.05); however, serum creatinine level was not increased (before: 1.99 ± 0.9 mg/dL vs. after: 1.82 ± 1.8 mg/dL,p>0.05). This study provides strong evidence that HDVD supplementation does not worsen renal function in DSPN patients with an eGFR greater than 15 mL/min/1.73 m² (60). It is well known that kidney function gradually declines with age; however, a clinical study of monthly intramuscular injection of HDVD in very elderly people with an average age of 89.6 years has attracted our attention. The results of this study showed no further decline in renal function after intervention (creatinine level, before: 97.38 ± 18.08 mg/dL vs. after: 101.12 ± 20.39 umol/L, p>0.05), and there was no significant change in blood calcium levels (before: 2.30 ± 0.14 mg/dL vs. after:2.34 ± 0.11 mmol/L, p>0.05) (61). Our study will also collect data on changes in renal function, blood calcium after HDVD.

Many researchers have explored whether increasing the dose of vitamin D supplementation will cause side effects or adverse reactions. In a study by Binkley et al. (37) administering 50000 units of vitamin D monthly to people aged > 65 years did not cause any toxicity or side effects. Another clinical study by Bian et al. (61) in people aged > 80 years showed that supplementation with 600000 units of vitamin D monthly for more than half a year might be safe. However, the sample size used in these studies was small. Therefore, we will use a monthly regimen of high-dose intramuscular vitamin D injections closely observed by the implementer to achieve better compliance and an earlier time to target. Additionally, our study will address the safety of monthly HDVD supplementation in individuals with insufficient vitamin D levels.

Although vitamins D₂ and D₃ have the same efficacy in exerting physiological effects, previous studies have suggested that vitamin D₂ supplementation may affect vitamin D₃ levels (56). This may be related to increased CYP24A1 enzyme activity in the liver, promoting the conversion of 25(OH)D₃ to 24,25-(OH)₂D₃, thereby inactivating it (57, 62). Additionally, 25(OH)D₂ and 25 (OH)D₃ compete for the binding to VDBP (63, 64). Notably, traditional immunoassays for vitamin D do not distinguish between 25(OH)D₂ and 25(OH)D₃, which may underestimate 25(OH)D₂ levels. Testing for vitamin D₂ can rule out the effects of sunlight (65). Therefore, we will use the gold standard LC-MS/MS method to distinguish between 25(OH)D₂ and 25 (OH)D₃ levels.

The strength of this study design is that it will determine whether an improvement in neuropathy accompanies an increase in vitamin D concentration due to HDVD supplementation. Notably, DSPN is associated with muscle atrophy; thus, decreasing the quality of life. Although previous studies suggested that vitamin D supplementation may improve muscle strength (52, 54, 66), our study will explore the effects of HDVD from multiple dimensions, such as quality of life and muscle strength. We will use the Short Form-12 (SF-12) questionnaire, comprising eight domains, to assess the quality of life. The SF-12 has satisfactory validity and reliability, showing good internal consistency in the psychological (Cronbach’s α=0.83) and physical component summaries (Cronbach’s α=0.81) (67). Moreover, we will measure the maximum grip strength of both hands before and 3 months post-intervention to assess changes in muscle strength.

This study design has some limitations. First, the trial will be conducted only in China’s population with T2DM and DSPN. Therefore, the results may not be directly applicable to other ethnic groups. Second, the latitudes of southern China will be selected for this study; therefore, the results may not be directly applicable to other latitudes. Nevertheless, the study can provide certain evidence that may require further verification in other regional or international multicenter studies. Third, owing to the difficulty in recruiting patients with DSPN, it will be difficult to maintain a balance between the groups. However, we will further correct factors, such as age, sex, and body mass index, through stratified analysis and by applying the analysis of covariance statistical strategy as far as possible.

In conclusion, this study will provide clinical evidence demonstrating the efficacy and safety of post-treatment HDVD supplementation for DSPN. Additionally, if HDVD supplementation can improve neuropathy, this study will provide new directions for future pharmacological research and clinical management of diabetic neuropathy.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the Ethics Committee of Longyan First Affiliated Hospital of Fujian Medical University (approval number: LYREC2022-013-01). The patients/participants provided their written informed consent to participate in this study.

TC, XX, PW, and MT participated in the study design. XX, PW, and MT supervised the study.TC prepared the first of the manuscript. TC and SL conducted the statistical strategy. LH performed sample size estimation. LH, XL, JC, SW, YZ, YL, LX, YQ, LQ, and YX edited and revised the manuscript. PW reviewed the methodology and the entire contents of the manuscript. All authors contributed to the article and approved the submitted version.