A total of 36 female specific pathogen-free (SPF) Lewis rats (weight, 120–140 g; age, 5–6 weeks) were purchased from Charles River Laboratories, Inc. Rats were maintained in SPF conditions at 23±2°C, 55±5% relative humidity, and under a 12-h light/dark cycle with ad libitum access to food and drinking water. Pentobarbital sodium (50 mg/kg) was injected intraperitoneally for anesthesia prior to modeling and repetitive nerve stimulation (RNS) detection to minimize animal pain. All experimental procedures were approved by the Experimental Animal Ethics Committee of Guangzhou University of Chinese Medicine (approval no. 20200509001; Guangzhou, China) and adhered to the National Institutes of Health Guide for the Care and Use of Animals guidelines (39).

Rat 97–116 peptide of the AChR-α subunit (R97-116, DGDFAIVKFTKVLLDYTGHI) was synthesized by GL Biochem (Shanghai) Ltd. Complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and sodium pentobarbital were purchased from MilliporeSigma (cat. nos. F5881, F5506 and P3761, respectively). PBS (cat. no. 10010-023) was purchased from Gibco (Thermo Fisher Scientific, Inc.). An AChR-antibody (AChR-Ab) assay kit (cat. no. ml003131) was obtained from Shanghai Enzyme-linked Biotechnology Co., Ltd. AS-IV (CAS no. 84687-43-4) was purchased from Chengdu Herbpurify Co., Ltd. A Hematoxylin and Eosin (H&E) Staining Kit (cat. no. C0105S) was purchased from Beyotime Institute of Biotechnology. ATPase and cytochrome c (Cyt-C) oxidase (COX) assay kits were obtained from Leagene; Beijing Regan Biotechnology Co., Ltd. (cat. nos. DE0045 and DE0031, respectively). Forward and reverse primers were purchased from Generay Biotech Co., Ltd. Phosphatase and tensin homolog-induced putative kinase 1 (PINK1), LC3I/II, Bcl-2, caspase 9 and GAPDH primary antibodies were purchased from Abcam (cat. nos. ab23707, ab128025, ab59348, ab52298 and ab8245, respectively). Parkin, p62, Bax, Cyt-C and caspase 3 primary antibodies were obtained from Cell Signaling Technology, Inc. (cat. nos. 4211, 39749, 2772, 11940 and 9662, respectively).

A total of 36 female Lewis rats were acclimated for 1 week in an SPF-level animal room, of which eight were randomly selected as the control group and the remaining 28 rats as the model group. The day of the first immunization was considered day 0. The modeling agent was prepared by mixing 100 µg R97-116 peptide, 0.1 ml CFA and 0.1 ml PBS evenly to form an emulsion. After the preparation of the emulsion mixture, four sites were selected for immunogen injection, including the back, abdomen, and left and right hind foot pads. Each site was injected with 50 µl immune emulsion and the injection site was washed with 75% ethanol. Each rat received a total of 200 µl modeling agent during the first immunization. The second and third immunization boosts were performed on days 30 and 45, respectively, after the first immunization. For these subsequent immunizations, the reagent was prepared by mixing 50 µg R97-116 peptide, 0.1 ml IFA and 0.1 ml PBS, which was injected subcutaneously at the same sites. During the second and third immunization periods, each rat received a total of 200 µl modeling agent each time. During each immunization period, for rats in the control group, the rats were injected subcutaneously with a mixed emulsion of 0.1 ml PBS and 0.1 ml CFA or IFA without R97-116 peptide, and the injection sites and doses were the same as those of the model group rats (40). During the modeling period, the mental state, activity, appetite, stool characteristics and hair luster of each group were recorded daily. The body weight of the rats was measured twice a week, and the Lennon score was recorded. The Lennon score was evaluated using the criteria described previously (41,42), and the assigned Lennon scores were as follows: Grade 0, no weakness; grade 1, fatigability or weakness only observed after exercise; grade 2, clinical signs of weakness present before exercise, including hunched posture or head down; grade 3, severe muscle weakness with dyspnea/apnea or moribund state; grade 4, death. Intermediate signs in rats were categorized into grades 0.5, 1.5, 2.5 or 3.5, accordingly. The model was evaluated 1 week after the third immunization. Rats in both groups undergoing experimental treatment were euthanized at day 80 or when they reached a humane endpoint, such as self-harming behavior, severe weight loss of 20%, compromised breathing or lack of response to external stimuli. Sodium pentobarbital (150 mg/kg) was injected intraperitoneally to euthanize the rats. Death was confirmed by monitoring the cessation of breathing and heartbeat, and pupil dilation. Throughout the course of the experiment, only one rat in the model group reached the predefined humane endpoints at the day 49.

After anesthesia, the rats in the model group were immobilized. Stimulating electrodes of the electrophysiological recorder (cat. no. PL3516; PowerLab; ADInstruments, Ltd.) were placed near the sciatic nerve in the gastrocnemius muscle, recording electrodes were subcutaneously implanted in the medial head of the gastrocnemius muscle and Achilles tendon on the same side, and reference electrodes were subcutaneously implanted in the abdomen (43). All electrodes received 10 pulses of 5 Hz electrical stimulation, and the percentage change of the first and fifth action potentials was measured. A positive result was defined as an attenuation rate of >10%.

On day 7 after modeling completion, rats were anesthetized with an intraperitoneal injection of 30 mg/kg pentobarbital sodium. Subsequently, a single blood sample of 0.2–0.4 ml was collected from the orbital venous plexus of each rat. Additionally, upon completion of the entire experiment and euthanasia of the rats, a blood sample of 0.8–1 ml was collected once from the abdominal aorta. The collected blood samples were placed in centrifuge tubes for 30 min and left to stand. The tubes were then centrifuged at 1,500 × g for 10 min at 4°C, and the supernatant was collected and stored at −80°C. The AChR-Ab levels in the rats were measured in the serum using the aforementioned specific ELISA kit.

Following three separate immunizations, 24 of the 28 rats in the model group were successfully modeled, and the rate at which the EAMG rat model was successfully established was 85.71%. The model group was randomly divided into three subgroups: Model group, low-dose AS-IV (L-ASIV) group and high-dose AS-IV (H-ASIV) group (n=8 rats/group). The rats in the L-ASIV and H-ASIV groups were treated with 40 and 80 mg/kg/day AS-IV by gavage, respectively (44–46). The control and model groups were administered equal volumes of saline by gavage, and the gavage volume was calculated according to the principle of 1 ml/kg body weight. During the four-week Drug intervention, rats in each group were administered via gavage once a day, following the respective treatment method. The rats were fasted for 24 h after the last administration. Sodium pentobarbital (150 mg/kg) was injected intraperitoneally for euthanasia, and death was confirmed by monitoring the cessation of breathing and heartbeat, and pupil dilation (47).

After the rats were sacrificed, the gastrocnemius muscle tissue was immediately removed and fixed overnight at room temperature in 4% paraformaldehyde and was then embedded in conventional paraffin. The tissues were cut into 4-µm sections, which were stained with 100% hematoxylin solution at room temperature for 2 min and were then differentiated in 1% hydrochloric acid ethanol for several seconds. After rinsing with water and dehydrating in a series of increasing concentrations of ethanol, the sections were stained with 100% eosin solution at room temperature for 1 min. The sections were then placed in xylene until they became transparent and were mounted with neutral gum. Images of the sections from each group were captured under an optical microscope at a magnification of ×400 (Olympus BX53; Olympus Corporation).

After 4 weeks of treatment, gastrocnemius muscle samples were cut to a volume of 1 mm³ and fixed with 2.5% glutaraldehyde at 4°C for 2–6 h. The sections were then rinsed in phosphoric acid buffer solution for 30 min (repeated six times), fixed with 1% osmic acid at 4°C for 1.5–2 h, and rinsed again in phosphoric acid buffer solution for 10 min (repeated three times). After dehydration in a series of increasing concentrations of ethanol, the tissue samples were embedded in epoxy resin monomer overnight at 37°C. An ultramicrotome (RM2016; Leica Microsystems GmbH) was used for sectioning (60 nm), and the samples were then stained with uranium dioxide acetate at room temperature for 12 min and with lead citrate at room temperature for 8 min. Finally, the ultrastructure was observed using TEM (Jem-1400; JEOL, Ltd.).

An ATPase staining kit was used for ATPase staining. Fresh gastrocnemius muscle tissues were embedded in a frozen section embedding agent and frozen at −20°C for further processing. The embedded tissues were then cut into 6 µm frozen sections and placed in distilled water. The sections were incubated in calcium salt solution at 37°C for 5 min, then incubated with ATPase incubation solution at 37°C for 40 min, followed by washing with distilled water. Subsequently, the sections were immersed in cobalt nitrate solution at 37°C for 5 min and then in distilled water. They were then fixed with 4% neutral formalin at room temperature for 2 min and rinsed with running water for 2 min. The sections were placed in a prepared sulfide working solution and incubated for 2 min. After washing with water, the sections were mounted with glycerol gelatin onto slides. Images were captured under a fluorescence microscope (Olympus Corporation) at ×50 magnification and the average optical density values of the images were calculated using ImagePro Plus (version 6.0; Media Cybernetics, Inc.).

The frozen sections of gastrocnemius muscle of rats were dehydrated in an increasing concentration gradient of ethanol (5 min at each concentration). The frozen sections were then soaked in xylene solution twice (10 min each), the excess liquid on the slide was removed with absorbent paper and the slides were sealed with neutral gum. Subsequently, DAB solution was added to the frozen sections and incubated at 37°C in the dark for 90 min, before the sections were washed with distilled water three times (3 sec each). The staining of frozen sections was observed under an optical microscope (EcliPse E100; Nikon Corporation).

Total RNA was obtained from the gastrocnemius muscle tissue samples of rats using TRNzol-A⁺ (Tiangen Biotech Co., Ltd.). cDNA was synthesized by RT using a PrimeScript™ RT MasterMix Kit (Takara Bio, Inc.). The expression of target genes was quantified by qPCR with TB Green™ Premix Ex Taq™ II (Takara Bio, Inc.) using a CFX96 Real-Time PCR System (Bio-Rad Laboratories, Inc.). The RT temperature protocol was 37°C for 15 min and 85°C for 5 sec. The thermocycling conditions for qPCR amplification were 95°C for 30 sec for initial denaturation; followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec and extension at 72°C for 20 sec, followed by a final extension at 72°C for 5 min. The primer sequences of all target genes are shown in Table I. Relative quantification was performed using the 2^−ΔΔCq method (48), with GAPDH as the reference gene.

Total protein was extracted from the gastrocnemius muscle tissues with RIPA lysis buffer (cat. no. KGP2100; Nanjing KeyGen Biotech Co., Ltd.) on ice, then centrifuged at 10,000 × g for 15 min at 4°C to collect the supernatants. A bicinchoninic acid assay (cat. no. KGP902; Nanjing KeyGen Biotech Co., Ltd.) was used to measure the protein concentration. Equal amounts of protein (30 µg) were loaded on 10 and 12% SDS-gels, resolved using SDS-PAGE and subsequently transferred to a PVDF membrane. Membranes were blocked using 5% skimmed milk (cat. no. 232100; Difco; BD Biosciences) at room temperature for 1 h and were then incubated overnight with primary antibodies at 4°C with agitation. The following primary antibodies were used: Anti-PINK1 (1:1,000), anti-LC3A/B (1:1,000), anti-Bcl-2 (1:1,000), anti-caspase 9 (1:1,000), anti-GAPDH (1:5,000), anti-Parkin (1:1,000), anti-SQSTM1/p62 (1:1,000), anti-caspase 3 (1:1,000), anti-Bax (1:1,000) and anti-Cyt-C (1:1,000). Subsequently, the membranes were washed and incubated with the secondary HRP-conjugated Goat Anti-Rabbit IgG antibody (1:3,000; cat. no. CW0103S; CW Biosciences) or Goat Anti-Mouse IgG antibody (1:3,000; cat. no. CW0102S; CW Biosciences) at room temperature for 1 h, and the protein bands were observed using a Western ECL Substrate kit (cat. no. 170-5061; Bio-Rad Laboratories, Inc.). Signals were visualized using a gel imaging system (Chemi Doc XRS+; Bio-Rad Laboratories, Inc.), and Image Lab software (version 6.0; Bio-Rad Laboratories, Inc.) was used for densitometric analysis. All experiments were repeated independently at least three times.

All data are presented as the mean ± standard deviation or as median and IQR. Graphs were generated using GraphPad Prism version 9 (Dotmatics). SPSS version 22.0 (IBM Corp.) was used for data processing. Statistical differences between two groups were determined using unpaired Student's t-test. Comparisons among more than two groups were carried out using one-way analysis of variance, followed by the Tukey's post hoc test. The nonparametric Mann-Whitney U test or Kruskal-Wallis test followed by Dunn's multiple comparisons test were used to analyze the Lennon score data. P<0.05 was considered to indicate a statistically significant difference.

Lewis rats were immunized by subcutaneously injecting peptide R97-116 as an antigen to induce EAMG (Fig. 1A); ~20 days after the first immunization, the majority of the rats in the model group exhibited a decrease in water and food consumption, weight loss and muscle weakness (increased Lennon score), compared with the rats in the control group (Fig. 1B and C). After the third booster immunization, one rat was sacrificed due to reaching humane endpoints, and the rest of the model group rats exhibited decreased activity, low and weak cries, dull and yellow fur, marked weight loss and muscle weakness (increased Lennon score); the differences between the model group and the control group were statistically significant (both P<0.01; Fig. 1B and C). After modeling, the rats were anesthetized and the changes in the response to RNS in each group were measured using electromyography. The attenuation of the first action potential amplitude and the fifth action potential amplitude of model rats was >10%, and the differences between the model group and the control group was statistically significant (P<0.01; Fig. 1D and E). ELISA was used to detect the levels of AChR-Ab in the rats. Compared with in the control group, the AChR-Ab content in the serum of rats in the model group was significantly increased (P<0.01; Fig. 1F).

After modeling, the different groups were treated with normal saline or AS-IV (Fig. 2A) for 4 weeks. In the model group, the weight and muscle strength of rats continued to decline after treatment with normal saline. After 4 weeks of treatment, compared with in the model group, the average weight of EAMG rats in the H-ASIV and L-ASIV groups significantly increased (P<0.01; Fig. 2B). The Lennon score also significantly decreased, indicating improved MG symptoms (P<0.01; Fig. 2C). Electromyography (EMG) was then used to observe the changes in the response to RNS in each group, and the levels of AChR-Ab in the blood samples from each group were measured. There was no significant change in the EMG amplitude in the control group, but the EMG amplitude attenuation rate in the model group was >10% compared with that in the control group (P<0.01; Fig. 2D and E). By contrast, the RNS amplitude attenuation of rats in the H-ASIV and L-ASIV groups recovered and was significantly reduced compared with that in the model group (P<0.01 and P<0.05, respectively; Fig. 2D and E). Compared with in the control group, the AChR-Ab levels of rats in the model group were significantly increased (P<0.01; Fig. 2F). By contrast, the AChR-Ab levels in the blood samples from the H-ASIV and L-ASIV groups were significantly reduced compared with those in the model group (P<0.01 and P<0.05, respectively; Fig. 2F). Taken together, these results suggested that AS-IV can improve the symptoms of EAMG in a rat model.

The muscle fibers of rats in the control group were tight, normal and orderly, with nuclei evenly distributed at the edge of the fibers (Fig. 3A). In the model group, muscle fiber atrophy, widening of the spaces between fibers, edema and degeneration, connective tissue formation and mass inflammatory cell infiltration were observed. Following treatment with AS-IV, both the low and high dose groups showed varying degrees of improvement, especially at the higher dose, and the muscle fiber morphology was similar to that observed in the control group (Fig. 3A).

TEM was used to observe changes in the muscle fiber arrangement and mitochondrial ultrastructure in the gastrocnemius muscle tissue of rats (Fig. 3B). In the control group, the gastrocnemius muscle tissue was tight, the muscle fibers were arranged in an orderly manner, the mitochondrial structure was complete and clear, and the cristae were visible. In the model group, most muscle fibers appeared broken, the number of mitochondria was notably reduced, and the mitochondrial structure was incomplete. In the L-ASIV group, the muscle fibers of rats appeared severely broken, some mitochondria were swollen, vacuolated and showed aggregation, but the number of mitochondria was higher than that in the model group. In the H-ASIV group, fewer broken muscle fibers were observed compared with in the model group, with visible cristae and complete mitochondrial structure. In addition, autophagosomes in the gastrocnemius muscle tissue were observed using TEM. In the model group, no autophagosomes were observed under TEM, whereas AS-IV treatment markedly increased the number of autophagosomes (Fig. 3B). Briefly, these results indicated that AS-IV notably improved the morphology of the gastrocnemius muscle, the ultrastructure of mitochondria and the dysregulated mitophagy in rats.

ATPase hydrolyzes ATP into ADP and phosphate, and releases energy for almost all essential cellular processes (49). In the gastrocnemius muscle of control rats, active site of ATPase staining is brown and black, and positive expression of ATPase was detected (Fig. 4A and B). In the model group, the intensity of staining was low and ATPase expression was significantly reduced compared with that in the control group, suggesting that ATPase activity was decreased (P<0.01). After treatment with AS-IV, the intensity of staining was higher, expression increased and ATPase activity was significantly increased in H-ASIV and L-ASIV groups compared with that in the model group (P<0.01 and P<0.05, respectively; Fig. 4A and B). In summary, these results suggested that AS-IV can improve ATPase activity and restore mitochondrial function.

COX is an enzyme present in mitochondria, which serves a crucial role in maintaining the normal structure and function of mitochondria (50). To evaluate COX activity, an optical microscope was used to observe the differences in COX staining in the gastrocnemius muscle tissues after intragastric treatment for 4 weeks. COX staining of frozen sections of gastrocnemius tissue in the control group showed that the myofibrils were polygonal and of equal size; the color at active part of COX staining was indicated by brown grain precipitation. Compared with in the control group, COX activity in the model group was lower with lighter brown grain precipitation in most positively stained areas, and irregularities in the size and shape of myofibrils was observed (Fig. 4C). In the L-ASIV group, COX activity was low and the positively stained area showed a lighter brown grain precipitation, accompanied with the myofibrils exhibited irregular shapes. There was a notable difference in the H-ASIV group when compared with the model group, which presented regularly shaped myofibrils and more intense staining (Fig. 4C). Compared with in the control group, the intensity of distribution (IOD)/area of COX activity in the model group was significantly reduced (P<0.05; Fig. 4D). By contrast, compared with in the model group, in the H-ASIV group, the IOD/area of COX activity was significantly increased (P<0.05; Fig. 4D). Therefore, these results suggested that AS-IV improved COX activity to preserve mitochondrial structure and function.

To further understand the mechanism of action of AS-IV, its effects on the mRNA and protein expression levels of members of the mitophagy-related signaling pathway were determined. As shown in Fig. 5A-D, compared with those in the control group, the mRNA expression levels of PINK1, Parkin and LC3II in the model group were decreased, whereas the mRNA expression levels of p62 were increased (all P<0.01). Compared with in the model group, the difference in the mRNA expression levels of PINK1, Parkin, p62 and LC3II in the L-ASIV group was not significant, whereas the mRNA expression levels of PINK1, Parkin and LC3II were significantly increased in the H-ASIV group (P<0.05 or P<0.01). Additionally, the mRNA expression levels of p62 were significantly decreased in the H-ASIV group (P<0.01).

The protein expression levels of the aforementioned molecules were further assessed using western blotting following AS-IV treatment. As shown in Fig. 6A-E, the protein expression levels of PINK1, Parkin, and LC3II/I in the model group were significantly lower than those in the control group, whereas the protein expression levels of p62 were significantly increased (all P<0.01). After treatment with AS-IV, compared with those in the model group, the protein expression levels of PINK1, Parkin, and LC3II/I in the H-ASIV group were significantly higher, and the expression levels of p62 were significantly decreased (P<0.05 or P<0.01). By contrast, there was no significant difference in the protein expression levels in the L-ASIV group. Taken together, these results suggested that AS-IV can promote PINK1/Parkin-mediated mitophagy in the skeletal muscle of EAMG rats.

Subsequently, we conducted further investigations to observe the impact of AS-IV on the mRNA and protein expression levels of mitochondrial apoptosis-related signaling pathway components. As shown in Fig. 7A-E, the mRNA expression levels of Bax, Cyt-C, caspase 3 and caspase 9 were significantly higher (P<0.01), and the mRNA expression levels of Bcl-2 were significantly lower (P<0.01), in the skeletal muscle tissues of the model group compared with in the control group. After 4 weeks of AS-IV treatment, the expression levels of Cyt-C, Bax, caspase 3 and caspase 9 were significantly lower in the L-ASIV and H-ASIV groups than those in the model group (all P<0.01; except for Cyt-C in the L-ASIV group, P<0.05). By contrast, Bcl-2 expression in the H-ASIV and L-ASIV groups was significantly higher than that in the model group (P<0.01).

Western blotting was used to detect the protein expression levels of the aforementioned genes. As shown in Fig. 8A-F, the protein expression levels of Cyt-C, Bax, caspase 3 and caspase 9 were significantly higher (all P<0.01), and the expression levels of Bcl-2 were significantly lower (P<0.01) in the skeletal muscle of the model group compared with those in the control group. After 4 weeks of AS-IV treatment, the expression levels of Cyt-C, Bax, caspase 3 and caspase 9 in the H-ASIV and L-ASIV groups were significantly lower than those in the model group (all P<0.01; except for caspase 3 and 9 in the L-ASIV group, P<0.05). In addition, the protein expression levels of Bcl-2 in the H-ASIV and L-ASIV groups were significantly higher than those in the model group (P<0.01). In summary, these results indicated that AS-IV can ameliorate the expression of mitochondrial apoptosis-associated proteins/genes in the skeletal muscle of EAMG rats.