Forty male Sprague-Dawley rats, body mass (BM) 200 ± 10 g, Liaoning Chang Sheng Biotechnology Co. Ltd., SCXK (Liao) 2020-0001), were habituated at (22 ± 2)˚C, in 55% ± 5% humidity and with a 12 h light/dark cycle (lights on at 8:00 and off at 20:00). After one week of accommodation, rats were randomly divided into the normal group, SQD model group (model group), SQD model + stilled water group (SW group) and SQD model + Sijunzi decoction (CM group), 10 rats each group. Animal care procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and every effort was made to minimize the animal suffering.

The SQD model was established and assessed according to our previous study [5] . Briefly, in 2 weeks, rats in the model, SW and CM groups were provided with feed and water ad libitum on odd days and fed only with cabbage on even days, meanwhile they were forced to swim to fatigue every day. The evaluation standards included emaciation (BM decrease) checked by the electronic scales, poor appetite (decreases of food and water intakes) detected in the metabolic cages, mental fatigue (motion distances and vertical times) recorded with the OFT-100 opening activity experiment system (Chengdu TME Technology Co, Ltd., Chengdu, China), and limb weakness checked with YLS-13A Grip Strength Meter for Rats and Mice (Jinan Yi Yan Technology Development Co., Ltd., Shandong, China). Evaluation of the SQD model was finished by an investigator who was blinded for groups.

Sijunzi decoction (Radix Ginseng 9 g, Rhizoma Atractylodis Macrocephalae 9 g, Poria 9 g and Radix Glycyrrhizae 6 g) was purchased from the First Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, and after being soaked and boiled in stilled water, the concentration of Sijunzi decoction was adjusted to 1.40 g/mL. The equivalent dose for a rat was converted from the clinical dose of an adult, which was given to rats in the CM group by gavage twice a day with an interval of 8 h for 2 weeks. Rats in the normal group were not treated, those in the model and SW groups were separately applied actions of intrgastric administration and given 2 mL SW in the same way as the CM group.

After rats were euthanized by inhaling carbon dioxide, soleus muscle was collected and cut into pieces of 1 mm³ at 4˚C and fixed with 2.5% glutaraldehyde in phosphate buffer. After dehydration in ethanol with graded concentrations, specimens were treated with propyene oxide and embedded in Epon. Ultrathin sections stained with uranyl acetate and lead citrate were examined under a transmission electron microscope (JEM-1 200EX; Jeol, Tokyo, Japan).

Soleus muscle was stored at 4% paraformaldehyde solution, and then embedded in paraffin and sectioned at 6 µm. The sections were stained with hematoxylin and eosin and examined by a light microscopy. The myonuclei were dyed blue, and 100 myofibers each group (10 fibers each rat) were chosen by the investigator described above to measure their myonuclear numbers, cross-section area (CSA) by Image Pro 6.0 (Bethesda, MD, USA), and then the MND size was calculated by CSA/the myonuclear number [6] [7] .

Simply, soleus muscle was quickly frozen in liquid nitrogen, embedded in optimal cutting temperature compound and then sectioned at 8 µm using a freezing CM1950 microtome (Leica, Wetzlar, Germany). The sections were fixed with 4% paraformaldehyde for 10 min, washed in PBS, and then blocked for 30 min in blocking buffer containing 5% bovine serum albumin at 37˚C. Sections were incubated with the the mouse anti-paired box gene 7 (Pax7) and rabbit anti-myogenic differentiation antigen (MyoD) (both were 1:200; Santa Cruz Biotechnlogy, sc-514352, sc-377460) overnight at 4˚C. Sections were washed 3 times in PBS and incubated for 5 min at room temperature (RT) in the blocking solution, and were incubated with the goat anti-mouse IgG and goat anti-rabbit IgG diluted in blocking solution for 90 min at RT, and the nuclei were stained with DAPI (0.3 µM). After incubation, sections were washed 3 times with PBS, and a drop of Alexa Fluor 488/594 secondary antibody mixture was added and incubated at RT in darkness for 1 h. Finally, sections were mounted with DAPI and fluorescence was visualized using an Olympus FV10i confocal microscope (Tokyo, Japan). The Pax7+ and MyoD+ nuclei in 100 myofibers each group (10 myofibers each rat) were counted in the blind way as described above.

Data were processed using SPSS 25.0 and expressed as mean ± standard deviation (SD). Analysis of variance was conducted to test differences between groups and p < 0.05 was considered statistically significant.

Table 1 and Figure 1 showed that all items in the model and SW groups were significantly less than those in the normal group, indicating that the SQD mode was successfully established and SW could not treat SQD. Compared with those in the model and SW groups, data in the CM group increased obviously, which suggested that Sijunzi decoction treated SQD.

Figure 2(A) was the typical structures of the sarcomeres in the normal group, where the Z and M lines were clear, and mitochondria were between the myofilaments. Figure 2(B) and Figure 2(C) were morphological changes in the model and SW group, where some Z and M lines were disorderly, and mitochondria were smaller; Figure 2(D) showed the sarcomeres of the CM group were basically normal, and the mitochondria were bigger than those in the model and SW groups.

Note: verse the normal group, ^ap < 0.05, ^aap < 0.01; verse the model group, ^bp < 0.05; verse the SW group, ^cp < 0.05, ^ccp < 0.01.

Table 2 and Figure 3 showed that, compared with those in the normal group, three items were all decreased significantly in the model and SW groups, and the myonuclear number was increased, CSA and the MND were reduced obviously in the CM group; Compared with those in the model and SW groups, the myonuclear number and CSA were increased markedly in the CM group.

Pax7 promotes proliferation of the MSCs, so the Pax7+ cell number is used to detect MSCs’ proliferation [8] . Figure 4 left and Table 3 showed that the Pax7+

Note: verse the normal group, ^ap < 0.05, ^aap < 0.01; verse the model group, ^bbp < 0.01; verse the SW group, ^ccp < 0.01.

Note: verse the normal group, ^ap < 0.05, ^aap < 0.01; verse the model group, ^bp < 0.05; verse the SW group, ^cp < 0.05, ^ccp < 0.01.

cell numbers in the later 3 groups were all greater than that in the normal group, and that in the CM group was higher than those in the model and SW groups. MyoD plays an important role in differentiation of the MSCs, therefore its positive cell number is used to assess MSCs’ differentiation [9] . Based on Figure 4 and Figure 5 right and Table 3, the MyoD+ cell number in the CM group was higher than those in the former 3 groups significantly.