Effects of Huoxue Chubi decoction (活血除痹汤) on protein kinase B-mammalian target of rapamycin autophagy pathway in scleroderma Balb/c model mice

doi:10.19852/j.cnki.jtcm.2025.02.005

Abstract

Abstract:

OBJECTIVE: To explore the mechanisms by which Huoxue Chubi decoction (活血除痹汤, HXCB) affects the protein kinase B (Akt)-mammalian target of rapamycin (mTOR) autophagy pathway in scleroderma Balb/c model mice.

METHODS: A scleroderma model was established in male Balb/c mice, followed by daily administration of HXCB (4.6, 2.3 and 1.15 g·kg^-1·d^-1) for 4 weeks. Bodyweight, epidermal and dermal thickness, dermal collagen levels, cutaneous reactive oxygen species (ROS) levels, Akt, Phosphorylated Akt (p-Akt), mTOR, Phosphorylated mTOR (p-mTOR), B-celllymphoma-2-interacting myosin-like coiled-coil protein 1 (Beclin-1) and microtubule-associated protein A/B-light chain 3 (LC3) protein and messenger ribonucleic acid (mRNA) expression were assessed.

RESULTS: HXCB treatment significantly reduced epidermal and dermal thickness, dermal collagen levels, ROS levels and the mRNA and protein expression of factors in the Akt-mTOR signaling pathway compared to the scleroderma model group. Conversely, mice body weight and autophagy factors Beclin-1 and LC3 were significantly increased in mice receiving HXCB treatment. Moreover, finally, ROS expression positively correlated with skin thickness, collagen contents and the mRNA expression levels of Akt, while the protein and mRNA expression levels of Akt-mTOR pathway-related factors were inversely correlated with the protein and mRNA expression of Beclin-1 and LC3.

CONCLUSION: HXCB can regulate autophagy by invigorating Qiand promoting blood circulation, thereby reducing blood stasis, facilitating new tissue generation, and contributing to scleroderma treatment. This effect may be attributed to the promotion of autophagy and enhancement of collagen degradation through the reduction of tissue oxidative stress elicited by HXCB.

Key words: autophagy, proto-oncogene proteins c-akt, TOR serine-threonine kinases, scleroderma, Huoxue Chubi decoction

CHEN Xi, QU Tiange, JIA Hui, DUAN Xingwu, LI Jianhong, ZHANG Kaihui, ZHANG Runtian, WANG Ruijie. Effects of Huoxue Chubi decoction (活血除痹汤) on protein kinase B-mammalian target of rapamycin autophagy pathway in scleroderma Balb/c model mice[J]. Journal of Traditional Chinese Medicine, 2025, 45(2): 303-310.

Figures/Tables 6

Table 1 Comparison of skin thickness, ROS and collagen levels in each group ($\bar{x}±s$)

Group	n	Skin thickness (μm)	ROS (IOD/mg)	Collagen level (IOD/μm²)
Control	7	333.91±29.44^a	1616.40±295.02^a	0.26±0.05^c
BLM	7	544.36±77.19	9311.22±1790.95	0.43±0.09
Colchicine	7	436.81±50.84^b	3495.15±1976.69^c	0.28±0.03^c
HXCB-H	7	443.82±42.65^b	4681.03±1020.88^c	0.28±0.04^c
HXCB-M	7	465.59±69.88	8346.29±4142.16	0.34±0.08
HXCB-L	7	485.71±75.95	8723.89±4255.77	0.36±0.05

Figure 1 Effect of HXCB on skin thickness in scleroderma mice A: control group (× 400); B: BLM group (× 400); C: Colchicine group (× 400); D: HXCB-H group (× 400); E: HXCB-M group (× 400); F: HXCB-L group (× 400). n = 7. The scleroderma model mice were received subcutaneous 0.1 mL injections of 400 μg/mL BLM at the center of the depilated area, and intervention began 4 weeks after model was established. Colchicine (0.13 mg·kg-1·d-1), HXCB-H (4.6 g·kg-1·d-1), HXCB-M (2.3 g·kg-1·d-1) and HXCB-L (1.15 g·kg-1·d-1) were used for gavage once per day for 33-60 d, and mice were euthanized on day 61. BLM: bleomycin; HXCB-H: high-dose Huoxue Chubi decoction; HXCB-M: middle-dose Huoxue Chubi decoction; HXCB-L: low-dose Huoxue Chubi decoction. Dyeing method of all pictures were the Hematoxylin and eosin.

Figure 2 Effect of HXCB on collagen levels in scleroderma mice A: control group (× 400); B: BLM group (× 400); C: Colchicine group (× 400); D: HXCB-H group (× 400); E: HXCB-M group (× 400); F: HXCB-L group (× 400). n = 7. The scleroderma model mice were received subcutaneous 0.1 mL injections of 400 μg/mL BLM at the center of the depilated area, and intervention began 4 weeks after model was established. Colchicine (0.13 mg·kg-1·d-1), HXCB-H (4.6 g·kg-1·d-1), HXCB-M (2.3 g·kg-1·d-1) and HXCB-L (1.15 g·kg-1·d-1) were used for gavage once per day for 33-60 d, and mice were euthanized on day 61. BLM: bleomycin; HXCB-H: high-dose Huoxue Chubi decoction; HXCB-M: middle-dose Huoxue Chubi decoction; HXCB-L: low-dose Huoxue Chubi decoction. Dyeing method of all pictures were the Masson-trichrome. Masson-trichrome staining sections showed that muscle fibers were red and collagen fibers were blue.

Figure 3 Protein relative expression of p-Akt/Akt, p-mTOR/mTOR and LC3, Beclin-1 in each group A: p-Akt/Akt expression level in the groups; B: p-mTOR/mTOR expression level in the groups; C: LC3 expression level in the groups; D: Beclin-1 expression level in the groups. n = 3. The scleroderma model mice were received subcutaneous 0.1 mL injections of 400 μg/mL BLM at the center of the depilated area, and intervention began 4 weeks after model was established. Colchicine (0.13 mg·kg-1·d-1), HXCB-H (4.6 g·kg-1·d-1), HXCB-M (2.3 g·kg-1·d-1) and HXCB-L (1.15 g·kg-1·d-1) were used for gavage once per day for 33-60 d, and mice were euthanized on day 61. BLM: bleomycin; HXCB-H: high-dose Huoxue Chubi decoction; HXCB-M: middle-dose Huoxue Chubi decoction; HXCB-L: low-dose Huoxue Chubi decoction. Akt: protein kinase B; mTOR: mammalian target of rapamycin; p-Akt: phosphorylated Akt; p-mTOR: phosphorylated mTOR; Beclin-1: B-celllymphoma-2-interacting myosin-like coiled-coil protein 1; LC3: microtubule-associated protein A/B-light chain 3. The single factor analysis of variance of completely randomized design was used for parameter comparison between groups, aP < 0.001, bP < 0.01 vs BLM group.

Table 2 mRNA relative expression of Akt-mTOR signals and LC3, Beclin-1 in each group ($\bar{x}±s$)

Group	n	Akt	mTOR	LC3	Beclin-1
Control	7	0.955±0.051^a	0.780±0.119^a	0.938±0.071^a	0.959±0.057^a
BLM	7	2.563±0.215	2.419±0.172	0.600±0.042	0.392±0.033
Colchicine	7	2.017±0.189^a	1.800±0.142^a	1.295±0.096^a	0.778±0.043^a
HXCB-H	7	0.812±0.059^a	0.449±0.030^a	1.824±0.046^a	2.563±0.208^a
HXCB-M	7	1.055±0.023^a	0.740±0.044^a	1.697±0.039^a	2.051±0.068^a
HXCB-L	7	1.184±0.065^a	0.891±0.051^a	1.693±0.064^a	0.690±0.035^a

Figure 4 Effect of HXCB on LC3 in scleroderma mice A: control group (× 400); B: BLM group (× 400); C: Colchicine group (× 400); D: HXCB-H group (× 400); E: HXCB-M group (× 400); F: HXCB-L group (× 400). n = 7. The scleroderma model mice were received subcutaneous 0.1 mL injections of 400 μg/mL BLM at the center of the depilated area, and intervention began 4 weeks after model was established. Colchicine (0.13 mg·kg-1·d-1), HXCB-H (4.6 g·kg-1·d-1), HXCB-M (2.3 g·kg-1·d-1) and HXCB-L (1.15 g·kg-1·d-1) were used for gavage once per day for 33-60 d, and mice were euthanized on day 61. LC3: microtubule-associated protein A/B-light chain 3; BLM: bleomycin; HXCB-H: high-dose Huoxue Chubi decoction; HXCB-M: middle-dose Huoxue Chubi decoction; HXCB-L: low-dose Huoxue Chubi decoction. Dyeing method of all pictures were the immunohistochemical.

References 20.

1.	Zhao B. China Clinical Dermatology. Jiangsu: Jiangsu Science and Technology Press, 2017: 878-85.
2.	Chen X, Zhang RT, Duan XW. Research progress in TCM treatment of scleroderma. Zhong Guo Ma Feng Pi Fu Bing Za Zhi 2013; 29: 331-3.
3.	Domenico DP, Rosa V, Luciana G. Defective autophagy in fibroblasts may contribute to fibrogenesis in autoimmune processes. Curr Pharm Design 2011; 17: 3878-87. DOI PMID
4.	Yang JH, Gao XG, Fu ZR. Role of autophagy in the mechanism of liver immune tolerance. Jie Fang Jun Yi Xue Za Zhi 2014; 39: 503-6.
5.	Zhao H, Wang Y, Qiu T, Liu W, Yao P. Autophagy, an important therapeutic target for pulmonary fibrosis diseases. Clini Chim Acta 2020; 502: 139-47.
6.	Zhou X, Liu CF, Lu JH, Zhu LB, Li M. 2-Methoxyestradiol inhibits hypoxia-induced scleroderma fibroblast collagen synthesis by phosphatidylinositol 3-kinase/Akt/mTOR signaling. Rheumatology 2018; 57: 1675-84.
7.	Ruth SS, Zvulun E. Regulation of autophagy by ROS: physiology and pathology. Trends Biochem Sci 2011; 36: 30-8.
8.	Lei LF, Yan C, Bo L. Beclin-1: autophagic regulator and therapeutic target in cancer. Int J Biochem Cell Biol 2013; 45: 921-4. DOI PMID
9.	Jiang YQ, Kou JY, Han XB, et al. ROS-dependent activation of autophagy through the PI3K/Akt/mTOR pathway is induced by hydroxysafflor yellow a-sonodynamic hherapy in THP-1 macrophages. Oxid Med Cell Longev 2017; 1: 1-16.
10.	Chen X, Zhang RT, Xia M, Duan XW. Introduction of Duan Xingwu's experience in treating localized scleroderma. Zhong Guo Zhong Xi Yi Jie He Pi Fu Xing Bing Xue Za Zhi 2015; 14: 164-6.
11.	Wang WY. Study on medication rules of Professor Duan Xingwu in the treatment of scleroderma based on data mining. Beijing: Beijing University of Chinese Medicine, 2021: 1-72.
12.	Qu X. Dermatology and venereology of Traditional Chinese Medicine. Beijing: China Traditional Chinese Medicine Press, 2009: 212-6.
13.	Duan XW. Interpretation of classic Chinese medical cases-dermatology. Beijing: China Medical Science and Technology Press, 2016: 198-212.
14.	Bogna GG, Mariusz P. Oxidative damage and antioxidative therapyin systemic sclerosis. Mediat Inflamm 2014; 9: 1-11.
15.	Jiang XW, Wang YC, Chang C. Regulation of autophagy by Traditional Chinese Medicine. Zhong Yi Za Zhi 2017; 58: 1566-8.
16.	Huang GH, Ji XY, Wu DL, et al. Relationship between autophagy and Qi deficiency and phlegm stasis in Traditional Chinese Medicine. Zhong Yi Za Zhi 2011; 52: 1717-19.
17.	Laplante M, Sabatini DM. mTOR signaling in growth control and disease. Cell 2012; 149: 274-93. DOI PMID
18.	Wesselborg S, Stork B. Autophagy signal transduction by ATG proteins: from hierarchies to networks. Cell Mol Life Sci 2015; 72: 4721-57. DOI PMID
19.	Deretic V. Autophagy in leukocytes and other cells: mechanisms, subsystem organization, selectivity and links to innate immunity. J Leukoc Biol 2016; 100: 969-78.
20.	Yu L, Chen Y, Tooze SA. Autophagy pathway: cellular and molecular mechanisms. Autophagy 2018; 14: 207-15. DOI PMID

[1]	SHI Jinyu, PAN Fuwei, GE Haiya, YANG Zongrui, ZHAN Hongsheng. Mechanism of Qigu capsule (芪骨胶囊) as a treatment for sarcopenia based on network pharmacology and experimental validation [J]. Journal of Traditional Chinese Medicine, 2025, 45(2): 399-407.
[2]	ZHANG Gedi, LIU Gengxin, GUO Min, LUO Fuli, YAN Ziyou, GE Wei. Effect of phosphatase and tensin homolog-induced putative kinase 1/ E3 ubiquitin ligase Parkin mediated mitochondrial autophagy on chronic kidney disease myocardial injury and the intervention mechanism of Shenshuai recipe (肾衰方) [J]. Journal of Traditional Chinese Medicine, 2024, 44(5): 934-943.
[3]	JIN Hong, WANG Xinna, WANG Ruonan, LI Jinjian, YU Junchao, ZHAO Dexi, ZHAI Lu. Neuroprotective effect of Naochuxue prescription (脑出血方) on intracerebral hemorrhage: inhibition of autophagy via downregulating high mobility group box-1 [J]. Journal of Traditional Chinese Medicine, 2024, 44(5): 944-953.
[4]	ZHENG Peng, MENG Ying, LIU Meijun, YU Di, LIU Huiying, WANG Fuchun, XU Xiaohong. Electroacupuncture inhibits hippocampal oxidative stress and autophagy in sleep-deprived rats through the protein kinase B and mechanistic target of rapamycin signaling pathway [J]. Journal of Traditional Chinese Medicine, 2024, 44(5): 974-980.
[5]	YU Siyun, ZHANG Shiwen, XIA Yu, LIU Xiaoqing, LIU Yajie, FU Jinrong. Network-based pharmacology and experimental validation to explore the mechanism of action of the Jiawei Pentongling formula (加味盆痛灵方) for the treatment of endometriosis-related pain [J]. Journal of Traditional Chinese Medicine, 2024, 44(5): 991-999.
[6]	ZHI Guoguo, SHAO Bingjie, ZHENG Tianyan, JI Shaoxiu, LI Jingwei, DANG Yanni, LIU Feng, WANG Dong. Efficacy of Ganshuang granules (肝爽颗粒) on non-alcoholic fatty liver and underlying mechanism: a network pharmacology and experimental verification [J]. Journal of Traditional Chinese Medicine, 2024, 44(1): 122-130.
[7]	LI Xi, LIN Xiangquan, CHEN Dongdong, LIU Hui. B-cell lymphoma-2 phosphorylation at Ser70 site-related autophagy mediates puerarin-inhibited the apoptosis of MC3T3-E1 cells during osteoblastogenesis [J]. Journal of Traditional Chinese Medicine, 2024, 44(1): 27-34.
[8]	YANG Xiaohui, WANG Jian, CHENG Li, ZHANG Yuxi, HUANG Jianlin, LIU Minghua. Active compounds of Caodoukou (Semen Alpinia Katsumadai) inhibit the migration, invasion and metastasis of human pancreatic cancer cells by targeting phosphoinosmde-3-kinase/ protein kinase B/mammalian target of rapamycin pathway [J]. Journal of Traditional Chinese Medicine, 2023, 43(5): 876-886.
[9]	GUO Zhaoan, SUN Lina, LIU Yingying, LI Ruifeng, LIU Chong, DIAO Ke, SHI Jing, SUN Jun. Qizhi Jiangtang capsule (芪蛭降糖胶囊) activates podocyte autophagy in diabetic kidney disease by inhibiting phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin pathways [J]. Journal of Traditional Chinese Medicine, 2023, 43(4): 667-675.
[10]	WU Haiyang, WANG Ying, HAN Wei, LI Huihui, JI Haisheng, LIU Xiuxiu. Protective effect of Tongdu Tiaoshen acupuncture combined with Xiaoxuming decoction (小续命汤) on dopaminergic neurons in Parkinson’s disease model [J]. Journal of Traditional Chinese Medicine, 2023, 43(3): 484-493.
[11]	YANG Mengzhe, ZHANG Beibei, LIANG Zhenqiang, CHENG Nannan, Lü Anqiao, YANG Jianyu, GUO Xingzhe, BAI Xianyu, HUANG Yuanjiao, JIAO Aijun, XU Ning. Sanguinarine suppresses cell proliferation, migration and invasion in nasopharyngeal carcinoma via inhibiting mTOR signaling [J]. Journal of Traditional Chinese Medicine, 2022, 42(5): 687-692.
[12]	ZHAO Lixia, SUN Wei, BAI Decheng. Protective effect of resveratrol on rat cardiomyocyte H9C2 cells injured by hypoxia/reoxygenation by regulating mitochondrial autophagy via PTEN-induced putative kinase protein 1/Parkinson disease protein 2 signaling pathway [J]. Journal of Traditional Chinese Medicine, 2022, 42(2): 176-186.
[13]	Jing QIAN, Xiaohong WANG, Benzhong WEI, Guoxiong ZHOU, Shunxing ZHU, Chun LIU. Therapeutic effects of salidroside vs pyrrolidine dithiocarbamate against severe acute pancreatitis in rats [J]. Journal of Traditional Chinese Medicine, 2022, 42(1): 49-57.
[14]	Xing DU, Tianlong LIU, Wendi TAO, Maoxing LI, Xiaolin LI, Lan YAN. Effect of aqueous extract of Astragalus membranaceus on behavioral cognition of rats living at high altitude [J]. Journal of Traditional Chinese Medicine, 2022, 42(1): 58-64.
[15]	Feng HAO, Qiang WANG, Lei LIU, Libin WU, Ronglin CAI, Jiajia SANG, Jun HU, Jie WANG, Qing YU, Lu HE, Yingchao SHEN, Yiming MIAO, Ling HU, Zijian WU. Effect of moxibustion on autophagy and the inflammatory response of synovial cells in rheumatoid arthritis model rats [J]. Journal of Traditional Chinese Medicine, 2022, 42(1): 73-82.

Home

Online First

Author Center

Reviewer Center

Issues

Announcement

About Us