Effects of Indirect Moxibustion on Skeletal Muscles in Mouse Model of Skeletal Muscle Adiposity

Article information

Acupunct. 2014;31(1):7-21
Publication date (electronic) : 2014 March 20
doi : https://doi.org/10.13045/acupunct.2014002
1Haneulche Oriental Medicine Clinic
2Nurijae Oriental Medicine Clinic
*Corresponding author : Nurijae Oriental Medicine Clinic, 5591, Doan-daero, Yuseong-gu, Daejeon, 305-301 Republic of Korea, Tel : 82-42-482-1075 E-mail : hkeacu@dju.kr
Received 2014 January 03; Revised 2014 March 04; Accepted 2014 March 07.

Abstract

Objectives:

To observe the regenerative effects of indirect moxibustion, a traditional Korean medical treatment on skeletal muscles using mouse model of skeletal muscle adiposity.

Methods:

Twenty seven ICR male mice were randomly assigned into Intact control(n=3), glycerol treatment together without moxibustion(n=12), and glycerol treatment together with moxibustion (n=12) groups. Mice of glycerol treatment groups were injected with 50 μl DW(distilled water) containing 50 % of glycerol into the two tibialis anterior.

After injection, moxibustion was applied at ‘Shenshu’(BL23) and ‘Zusanli’(ST36) acupoints three times per each session, every days for twelve days(total 12 treatments).

Phospho-Erk1/2, Myostatin protein levels were analyzed by western blotting and immunofluorescence staining techniques for tissues of the tibialis anterior muscle. Smad, phospho-Smad were analyzed by immunofluorescence staining.

Results:

  1. Histological analysis of sections from injected TA muscles showed that glycerol induced rapidly muscle necrosis, with a maximum at day 3. 6 days and 9 days after injection, muscle was regenerating.

  2. According to western blotting and immunofluorescence staining, phospho-Erk1/2 protein signals in glycerol treatment with moxibustion group were stronger compared to Intact and glycerol treatment without moxibustion group.

  3. According to western blotting and immunofluorescence staining, myostatin protein signals in glycerol treatment without moxibustion group were stronger compared to Intact and glycerol treatment with moxibustion group.

  4. According to immunofluorescence staining, Smad protein signals in glycerol treatment without moxibustion group were stronger compared to Intact and glycerol treatment with moxibustion group.

  5. According to immunofluorescence staining, phospho-Smad protein signals in glycerol treatment without moxibustion group were stronger compared to Intact and glycerol treatment with moxibustion group.

Conclusions :

These results confirm that indirect moxibustion of ‘Shenshu’(BL23) and ‘Zusanli’(ST36) influences muscle regeneration in mouse models of skeletal muscle adiposity. Further discussion, and the establishment of moxibustion mechanism will prompt clinical application of moxibustion.

Fig. 1.

Hemotoxylin and eosin(H & E) staining of tibialis anterior(TA)

After glycerol administration, muscle tissue sections were stained, and histologically analyzed by bright field microscopy.

Fig. 2.

High power images of H & E stained sections

Resolution of muscle cells and appearance of small cells were noted at 3 days after glycerol treatment followed by muscle tissue recovery at later time points.

Fig. 3.

Comparison in myostatin-staining intensity of normal and glycerol-treated muscle tissue 6 days later

Muscle sections were used for immunofluorescence staining to detect myostatin protein, and nuclear staining to identify individual cells.

Fig. 4.

Quantitation of phospho-Erk1/2 in the muscle

Protein lysates from the muscles tissues treated as indicated in the Figure were analyzed by western blot analysis.

a) : intact.

b) : glycerol treatment with moxibustion.

c) : glycerol treatment without moxibustion.

Fig. 5.

Immunofluorescence staining of phospho-Erk1/2 in the muscle tissue

Sections of skeletal muscle were used for immunostaining for phospho-Erk1/2.

a) : glycerol treatment without moxibustion.

b) : glycerol treatment with moxibustion.

Fig. 6.

Differential localization of phospho-Erk1/2 with individual nuclei

The nuclei in the muscle tissue were identified by Hoechst nuclear staining. Sections from glycerol treatment together with moxibustion(Moxi) or without moxibustion(Non-Moxi) were analyzed.

a) : glycerol treatment with moxibustion.

b) : glycerol treatment without moxibustion.

Fig. 7.

Quantitation of myostatin protein in the muscle tissue given different treatments

Protein lysates from the muscles tissues treated as indicated in the Figure were analyzed by western blot analysis.

a) : intact.

b) : glycerol treatment with moxibustion.

c) : glycerol treatment without moxibustion.

Fig. 8.

Immunofluorescence staining of myo statin in the muscle tissue

Sections of skeletal muscle were used for immunostaining for Myostatin. Myostatin signals were seen in green.

a) : glycerol treatment without moxibustion.

b) : glycerol treatment with moxibustion.

Fig. 9.

Immunofluorescence staining of Smad in the muscle tissue

Sections of skeletal muscle were used for immunostaining for Smad. Smad signals were seen in red.

a) : glycerol treatment without moxibustion.

b) : glycerol treatment with moxibustion.

Fig. 10.

Colocalization analysis of Smad and phospho-Erk1/2 in the muscle tissue

Merged images for Smad and phospho-Erk1/2 indicates colocalized signals in yellow. Merged images of Smad with Hoechst-stained nuclei reveal that they were not mostly colocalized. The sections were prepared 6 days after glycerol treatment together with moxibustion.

Fig. 11.

Colocalization analysis of Smad and phospho-Erk1/2 in the muscle tissue

Merged images for Smad and phospho-Erk1/2 indicates colocalized signals in yellow. Merged images of Smad and phospho-Erk1/2 with Hoechst-stained nuclei reveal that they were colocalized to some extent. The sections were prepared 12 days after glycerol treatment together with moxibustion.

Fig. 12.

Colocalization analysis of Smad and phospho-Erk1/2 in the muscle tissue

Merged images for Smad and phospho-Erk1/2 indicates colocalized signals in yellow. Merged images of Smad and phospho-Erk1/2 with Hoechst-stained nuclei reveal that they were highly colocalized in an area where the nuclei were heavily localized. The sections were prepared 6 days after glycerol treatment together with moxibustion.

Fig. 13.

Immunofluorescence staining of phosph o-Smad in the muscle tissue

Sections of skeletal muscle were used for immunostaining for phospho-Smad. phospho-Smad signals were seen in red.

a) : gycerol treatment without moxibustion.

b) : gycerol treatment with moxibustion.

Fig. 14.

Colocalization analysis of myostatin and phospho-Smad in the muscle tissue

Merged images for myostatin and phospho-Smad indicates colocalized signals in yellow. In moxibustion group, signal intensity of two proteins are low, and much stronger signals were observed in non-moxibustion group.

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Article information Continued

Fig. 1.

Hemotoxylin and eosin(H & E) staining of tibialis anterior(TA)

After glycerol administration, muscle tissue sections were stained, and histologically analyzed by bright field microscopy.

Fig. 2.

High power images of H & E stained sections

Resolution of muscle cells and appearance of small cells were noted at 3 days after glycerol treatment followed by muscle tissue recovery at later time points.

Fig. 3.

Comparison in myostatin-staining intensity of normal and glycerol-treated muscle tissue 6 days later

Muscle sections were used for immunofluorescence staining to detect myostatin protein, and nuclear staining to identify individual cells.

Fig. 4.

Quantitation of phospho-Erk1/2 in the muscle

Protein lysates from the muscles tissues treated as indicated in the Figure were analyzed by western blot analysis.

a) : intact.

b) : glycerol treatment with moxibustion.

c) : glycerol treatment without moxibustion.

Fig. 5.

Immunofluorescence staining of phospho-Erk1/2 in the muscle tissue

Sections of skeletal muscle were used for immunostaining for phospho-Erk1/2.

a) : glycerol treatment without moxibustion.

b) : glycerol treatment with moxibustion.

Fig. 6.

Differential localization of phospho-Erk1/2 with individual nuclei

The nuclei in the muscle tissue were identified by Hoechst nuclear staining. Sections from glycerol treatment together with moxibustion(Moxi) or without moxibustion(Non-Moxi) were analyzed.

a) : glycerol treatment with moxibustion.

b) : glycerol treatment without moxibustion.

Fig. 7.

Quantitation of myostatin protein in the muscle tissue given different treatments

Protein lysates from the muscles tissues treated as indicated in the Figure were analyzed by western blot analysis.

a) : intact.

b) : glycerol treatment with moxibustion.

c) : glycerol treatment without moxibustion.

Fig. 8.

Immunofluorescence staining of myo statin in the muscle tissue

Sections of skeletal muscle were used for immunostaining for Myostatin. Myostatin signals were seen in green.

a) : glycerol treatment without moxibustion.

b) : glycerol treatment with moxibustion.

Fig. 9.

Immunofluorescence staining of Smad in the muscle tissue

Sections of skeletal muscle were used for immunostaining for Smad. Smad signals were seen in red.

a) : glycerol treatment without moxibustion.

b) : glycerol treatment with moxibustion.

Fig. 10.

Colocalization analysis of Smad and phospho-Erk1/2 in the muscle tissue

Merged images for Smad and phospho-Erk1/2 indicates colocalized signals in yellow. Merged images of Smad with Hoechst-stained nuclei reveal that they were not mostly colocalized. The sections were prepared 6 days after glycerol treatment together with moxibustion.

Fig. 11.

Colocalization analysis of Smad and phospho-Erk1/2 in the muscle tissue

Merged images for Smad and phospho-Erk1/2 indicates colocalized signals in yellow. Merged images of Smad and phospho-Erk1/2 with Hoechst-stained nuclei reveal that they were colocalized to some extent. The sections were prepared 12 days after glycerol treatment together with moxibustion.

Fig. 12.

Colocalization analysis of Smad and phospho-Erk1/2 in the muscle tissue

Merged images for Smad and phospho-Erk1/2 indicates colocalized signals in yellow. Merged images of Smad and phospho-Erk1/2 with Hoechst-stained nuclei reveal that they were highly colocalized in an area where the nuclei were heavily localized. The sections were prepared 6 days after glycerol treatment together with moxibustion.

Fig. 13.

Immunofluorescence staining of phosph o-Smad in the muscle tissue

Sections of skeletal muscle were used for immunostaining for phospho-Smad. phospho-Smad signals were seen in red.

a) : gycerol treatment without moxibustion.

b) : gycerol treatment with moxibustion.

Fig. 14.

Colocalization analysis of myostatin and phospho-Smad in the muscle tissue

Merged images for myostatin and phospho-Smad indicates colocalized signals in yellow. In moxibustion group, signal intensity of two proteins are low, and much stronger signals were observed in non-moxibustion group.