2.1. Human Muscle Tissue Sample Collection

All experiments of this study were performed in accordance with the guidelines of the Ethics Committee on Human and Animal Experiments (Huashan Hospital, Fudan University). Human atrophic and normal muscles were obtained from three donors diagnosed with upper trunk BPI (brachial plexus injury). Inclusion criteria were as follows: (1) upper trunk BPI 6 months ago diagnosed by preoperative physical examination and electromyography; (2) uninjured accessory nerve and second to fourth cervical nerve; (3) 18–50 years old; (4) no other diseases; and (5) willing to participate in this study. Pieces of denervated deltoid muscle and normal (non-denervated) sternocleidomastoid muscle about 2 cm in length and 1 cm in diameter were resected from each patient during surgery (under intravenous anesthesia with intubation) and preserved in 4% paraformaldehyde until use. Information of collected patients is listed in Supplementary Table 1.

2.2. Experimental Animals

Male C57BL/6 mice aged 6–8 weeks and weighing 22–25 g were purchased from the Vital River Laboratory Animal Technology Co. (Zhejiang, China). The mice were housed in standard cages in a room at 23°C and 50% relative humidity on a 12-h: 12-h light/dark cycle. The study was approved by the ethics committee of Fudan University (202107012S, Date: 2021/7/30). The whole experiment was conducted from 2021/8/1 to 2023/7/30.

2.3. Experimental Grouping

Mice in all denervation groups were subjected to unilateral sciatic nerve transection under anesthesia (each animal was anesthetized by an intraperitoneal injection of 50 mg/kg 1% sodium pentobarbital), as previously described [21]. In brief, after deep anesthetization by intraperitoneal injection with sodium pentobarbital 50 mg/kg, a 0.5 cm-long sciatic nerve portion in the right hind leg of the mouse was resected, the two nerve ends were buried in muscle, and the incision was closed using 4 − 0 absorbable sutures. After sciatic nerve transection, mice in the Gap-19 group were treated with Gap-19 every 2 d via tail vein injection [22]. After sciatic nerve transection, mice in the blueberry group were treated daily with blueberry extracts dissolved in 37°C distilled water by intragastric administration by gastric tube. The mice in the denervation group received the same amount of distilled water daily. The mice were sacrificed after materials were obtained by overdose intraperitoneal injection with sodium pentobarbital 180 mg/kg.

Operations were all done at 9–11 am. Also, operations and tissues collecting procedures were followed in the same order. We have to state that we did not control cage location and room brightness, which were under unified management in Fudan University.

2.4. Wet Weight

Seven days or fourteen days after denervation, with and without blueberry extract administration, the mice were anesthetized, and the gastrocnemius muscles of both the left and right hind legs were removed, washed with saline, and weighed. The loss of muscle weight ratio was defined as the weight of the contralateral side minus the muscle weight of the nerve injury side, divided by the weight of the contralateral side. The muscle samples were stored in 4% paraformaldehyde at −80°C until use.

2.5. Grip Strength

Grip strength meter was used to measure the hindlimb grip strength 14 d after operation. Each mouse was allowed to grasp the machine with their forelimb taped to the chest, and then its tail was gently pulled back and the tension was recorded automatically by the machine. Each mouse had three times to perform the test, and 30°s rest was given between each test. The remainder values were recorded and normalized with whole body weight for analysis [23].

2.6. Hematoxylin and Eosin (HE)

Gastrocnemius muscle samples from mice and patients were fixed in 4% paraformaldehyde in room temperature and embedded in paraffin. The samples were cut at a thickness of 5 μm. Before staining, the slices were dewaxed by xylene in room temperature for 2-3 min until the wax was completely dissolved. Then, orderly move the slices into 100%, 90%, 80%, and 70% ethanol, water, and distilled water for about 2 min. The sections were stained with hematoxylin and eosin (HE) (Beyotime, Shanghai, China) to evaluate histopathologic changes. An EclipseCi-L camera light microscope was used to select the target area of the tissue for 400x imaging so that the tissue could fill the whole field of view as much as possible to ensure that the background light of each photo was consistent. The mean diameter of myofibers was determined by blinded analysis using ImageJ software (National Institutes of Health, Bethesda, MD, United States) from five randomly captured images per mouse in each experimental condition [24].

2.7. Immunofluorescence

To reveal Akt and Cx43 proteins in denervated muscles, double immunostaining was performed. The sections were deparaffinized with xylene in room temperature for 2-3 min until the wax was completely dissolved and rehydrated with ethanol, and antigen retrieval was performed in a 0.01-M citrate buffer (pH, 6.0) in a 95°C pressure cooker for 10 min, followed by natural cooling to room temperature. The paraformaldehyde-fixed denervated muscle was incubated with the primary antibody, followed by the secondary antibody, and subsequently mounted with DAPI. The primary antibodies used were rabbit anti-Akt (1 : 100, cat. no. AF1789, Beyotime Institute of Biotechnology Co. Ltd.) and rabbit anti-Cx43 (1 : 100, cat. no. 52559, Cell Signaling Technology, Danvers, MA, USA). The primary antibodies were incubated in 37°C for 1 h. Samples were viewed using a fluorescence microscope at 400x magnification and analyzed by Huygens software.

2.8. Western Blot Analysis

Frozen gastrocnemius muscle samples were homogenized in radioimmunoprecipitation assay buffer containing 1 mM phenylmethylsulfonyl fluoride and a Protease Inhibitor Cocktail (Roche Applied Science). Lysates were centrifuged for 20 min at 12, 000 × g (4°C) and the protein level in the supernatant was quantified using a bicinchoninic acid assay kit (Beyotime). Proteins were separated by SDS-PAGE (Beyotime) and transferred to a polyvinylidene difluoride membrane (Beyotime), which was blocked with 5% non-fat dry milk in Tris-buffered saline at room temperature for 1 h, followed by incubation at 4°C for 20 h with primary antibodies: rabbit anti-Akt (1 : 1000, cat. no. AF1789, Beyotime Institute of Biotechnology Co. Ltd.), rabbit anti-p-Akt (1 : 1000, cat. no. ab38449, Abcam), rabbit anti-Cx43 (1 : 5000, cat. no. C6219, Sigma), and rabbit anti-p-Cx43 (1 : 1000; cat. no. PA5-104820, Invitrogen). After three washes by TBST, the membrane was incubated with the appropriate secondary antibody (Abcam) at room temperature for 1 h. An enhanced chemiluminescence detection reagent and X-ray film were used for protein visualization.

2.9. Quantitative Real-Time PCR (qPCR)

An RNeasy kit (Qiagen, Valencia, CA, United States) was used to extract total RNA from the gastrocnemius muscle. cDNA was synthesized using a first-strand cDNA synthesis kit with oligo dT primers (Invitrogen, Carlsbad, CA, United States) and used for quantitative real-time PCR (qPCR). The thermal cycling conditions were as follows: 94°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 45 s, and 72°C for 1 min; and 72°C for 5 min. The relative expression levels of target genes were calculated using the cycle threshold (Ct) value. Akt and Cx43 expression levels were normalized to those of β-actin. We used Primer3 to design all primer sequences. The primer sequences were as follows: AKT: F, 5′ GAA CGA TGG CAC CTT TAT T 3′, R, 5′ AGT TGT TGA GTG GGG ACT CT 3′; Cx43: F, 5′ GAA AGG CGT GAG GGA AGT A 3′, R, 5′ TGA AGT CAA AGG AAA AGG AGA G 3′; β-actin: F, 5′-TCA TCA CTA TTG GCA ACG AGC-3′ and R, 5′-AAC AGT CCG CCT AGA AGC AC-3′.

2.10. Statistical Analysis of Data

All data are presented as mean ± SD. All statistical tests were performed using one-way t-test or ANOVA. All statistical analyses were conducted using SPSS software (version 17.0; SPSS Inc., Chicago, IL, United States). Statistical significance was set at p < 0.05.

3.1. Denervation Leads to Increased Expression of Cx43 and Decreased Expression of Akt in Atrophied Skeletal Muscle of Human Body

The HE staining also showed obvious atrophy of deltoid at 6 months postoperatively in histological level (Figure 1(a)). Compared with the sternocleidomastoid muscle, denervation induced increased expression of Cx43 in deltoid muscle, which was shown by both immunohistochemistry and immunofluorescence (Figures 1(b) and 1(c)). While, denervation induced decreased expression of Akt in deltoid muscle (Figures 1(d) and 1(e)). These results showed that the number of Cx43 increased and the number of Akt decreased after denervation.

3.2. Denervation Leads to Increased Expression and Activated Function of Cx43 in Atrophied Skeletal Muscle

The unilateral sciatic nerve was transected in mice in the denervated group. The wet weight ratio and grip strength decreased significantly at 14 d postoperatively (Figure 2(a)). The ipsilateral gastrocnemius also showed obvious atrophy at 14 d postoperatively in gross and histological examinations (Figures 2(a) and 2(b)). All these changes at 14 d were more obvious than those at 7 d (Supplementary Figures 1(A)–1(C)), and materials at 14 d postoperatively were used for further study. Compared with the control group, denervation induced increased expression of Cx43 in skeletal muscle cells at the level of transcription (Figures 2(c) and 2(d)) level. These results showed that the number of Cx43 increased after denervation. To explore if their function was activated, p-Cx43/Cx43 was analyzed by western blotting and it was found that p-Cx43/Cx43 increased continuously after denervation until 14 d (Figure 2(e)).

3.3. Gap-19 Inhibited the Function of Cx43 and Attenuated Denervated Muscle Atrophy

To determine the effects of Cx43 on skeletal muscle cells after denervation, we administered Gap-19 by tail intravenous administration, a selective Cx43 inhibitor, in mice in the Gap-19 group. Gap-19 reduced Cx43 expression in denervated skeletal gastrocnemius muscle during translation (Figures 3(b) and 3(c)) level and inhibited Cx43 activation (Figure 3(c)). In terms of histology (Figure 3(d)) and gross levels (Figure 3(a)), Gap-19 attenuated skeletal muscle atrophy 14 d after denervation. Gap-19 showed no obvious effects on skeletal muscle without denervation (Figure 3(e)). Combined with previous results, we conclude that Cx43 activation promotes denervation-induced skeletal muscle atrophy. Also, Gap-19 reduced denervation-induced Cx43 expression increase in skeletal gastrocnemius muscle during transcription level (Figure 4(f)).

3.4. Blueberry Extracts Attenuated Denervated Muscle Atrophy and Downregulated Cx43 Expression

Mice in the blueberry groups were treated daily with blueberry extracts dissolved in 37°C distilled water by intragastric administration from the day of operation. Figure 4(a) and Figure 4(b) show that, compared with gastrocnemius in denervation group (Figure 4(a) and 4(b) right), 200mg/kg (Figure 4(a) left), and 400 mg/kg (Figure 4(b) left) blueberry extracts treatment could improve the muscle mass of denervated gastrocnemius. Wet weight ratio comparison (Figure 4(c) also showed that denervation-induced skeletal muscle atrophy could be partially reversed by both doses of blueberry extracts treatment. Additionally, in histology (Figure 4(d)), skeletal muscle tissue showed a larger stained area of myocytes and a narrower intercellular space in both blueberry groups. Moreover, higher blueberry extract doses showed stronger protective effects. Next, we explored whether Cx43 was involved in the protective role of blueberry extracts against denervated muscle atrophy. Cx43 expression and p-Cx43/Cx43 were upregulated in denervated skeletal muscle, whereas blueberry extracts downregulated Cx43 expression in both transcriptions (Figure 4(f)) and translation (Figure 4(e)), and p-Cx43/Cx43 (Figure 4(e)) also decreased in the blueberry 200 group. However, the p-Cx43/Cx43 ratio did not decrease significantly in the blueberry 400 group.

3.5. Cx43/Akt Pathway Was Involved in the Effect of Blueberry Extracts on Denervated Muscle Atrophy

We then explored whether Akt contributes to the protective effect of blueberries and whether Cx43 interacts with Akt. HE staining (Figure 5(a)) and immunofluorescence (Figure 5(b)) showed that Gap-19-upregulated Akt expression was downregulated in denervated skeletal muscle tissue compared with the control group, which was upregulated by Gap-19. To explore whether Akt was involved in the protective effects of blueberry extracts on denervated skeletal muscle, we used western blot analysis and found that Akt expression was upregulated in the blueberry group compared to the denervation group (Figure 5(c)). Meanwhile, p-Akt expression was also upregulated in the blueberry group compared to the denervation group, which showed that activity of Akt was also restored in the blueberry group. Additionally, the relative Akt gene expression was downregulated in the denervation group, whereas Gap-19 and blueberry extracts reversed this change and even upregulated Akt expression compared to the control at the transcription level (Figure 5(d)). These results demonstrated that the Cx43/Akt interaction is involved in the effect of blueberry extracts on denervated muscle atrophy.