Low-intensity pulsed ultrasound relieved the diabetic peripheral neuropathy in mice via anti-oxidative stress mechanism

Diabetic peripheral neuropathy (DPN), as one of the most prevalent complications of diabetes, leads to significant pain and financial burden to patients. Currently, there was no effective treatment for DPN since the glucose control was just a prevention and the drug therapy only relieved the DPN pain. As a non-invasive physical therapy, low-intensity pulsed ultrasound (LIPUS) is utilized in the musculoskeletal and nerve injuries therapy. Studies revealed that LIPUS could regenerate nerves by the mechanical stimulation via oxidative stress pathway, which was thought as the important factor for DPN, and might have potential in the DPN therapy. This study aimed to identify a new therapeutic strategy for DPN using LIPUS. We analyzed the therapy effect and explored the therapeutic mechanism of LIPUS on DPN in mice.

This study involved animal experiments and C57BL/6J mice were randomly assigned to DPN model and Sham groups. The DPN model group was fed a high-fat chow diet and injected with streptozotocin (STZ) for 3 consecutive days (40 mg/kg/d), whereas the Sham group was fed a normal diet and injected with an equal volume of sodium citrate buffer. After the DPN model confirmed with the 84-day modeling process, the DPN mice were randomly allocated into the DPN group and the LIPUS group. The LIPUS group underwent ultrasound treatments with a center frequency of 1 MHz, a duty cycle of 20 %, and a spatial average temporal average intensity (I_SATA) of 200 $mW/{cm}^2$ for 20 min/d, 5 d/w. After the 56-day treatment, all mice were euthanized. LIPUS therapeutic effects were evaluated through measurements of fasting blood glucose (FBG), behavioral tests, oxidative stress tests, morphological analysis, immunofluorescence, and western blot analysis.

The results indicated that DPN mice had significantly higher FBG levels (28.77 ± 2.95 mmol/L) compared with sham mice (10.31 ± 1.49 mmol/L). Additionally, DPN mice had significantly lower mechanical threshold (4.13 ± 0.92 g) and higher thermal latency (16.20 ± 2.39 s) compared with the sham mice (7.31 ± 0.83 g, 11.67 ± 1.21 s). After receiving LIPUS treatment, the glucose tolerance tests (GTT) suggested that LIPUS treatment improved glucose tolerance, which was shown by a decrease in the area under the curve (AUC) for glucose in the LIPUS group (AUC = 2452 ± 459.33 min*mmol/L) compared with the DPN group (AUC = 3271 ± 420.90 min*mmol/L). Behavioral tests showed that LIPUS treatment significantly alleviated DPN-induced abnormalities by improving the mechanical threshold from 2.79 ± 0.79 g in the DPN group to 5.50 ± 1.00 g in the LIPUS group, and significantly decreasing thermal latency from 12.38 ± 1.88 s in the DPN group to 9.49 ± 2.31 s in the LIPUS group. Morphological observations revealed that DPN mice had a thinning and irregularly shaped myelin sheath, with 61.04 ± 5.60 % of abnormal nerve fibers in the sciatic nerve in LIPUS group, compared with 49.76 ± 4.88 % of abnormal nerve fibers in the LIPUS-treated group. Additionally, LIPUS treatment increased the mean fluorescence intensity of the associated nerve regeneration protein (i.e., Nf200) from 27.81 ± 0.32 arbitrary units in the DPN group to 37.62 ± 0.36 arbitrary units in the LIPUS group. Western blot and immunofluorescence analysis showed that LIPUS treatment significantly reduced Keap1 expression to 0.04 ± 0.06 relative units, compared with 0.17 ± 0.30 in the DPN group. Furthermore, immunofluorescence analysis revealed that LIPUS treatment promoted the production of its downstream antioxidant protein, heme oxygenase-1 (HO-1), with an increase in the fluorescence intensity from 27.81 ± 0.32 arbitrary units in the DPN group to 37.62 ± 0.36 arbitrary units in the LIPUS-treated group. The fluorescence intensity of Nrf2 was significantly higher in the LIPUS group, increasing from 4.90 ± 0.25 arbitrary units in the DPN group to 15.18 ± 2.13 arbitrary units in the LIPUS-treated group. Additionally, the malondialdehyde (MDA) levels, an indicator of oxidative stress, were significantly reduced in the serum, from 5.40 ± 0.48 nmol/ml in the DPN group to 4.64 ± 0.16 nmol/ml in the LIPUS-treated group, and in the sciatic nerve, from 16.17 ± 5.88 nmol/mg protein to 4.67 ± 2.10 nmol/mg protein, suggesting the oxidative stress was inhibited by LIPUS.

This study demonstrated for the first time that LIPUS could relive DPN through anti-oxidative stress process. This study suggests that LIPUS might be a new therapy strategy for DPN.