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

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.

1. INTRODUCTION

Scleroderma, marked by the progressive hardening of collagen fibers in the skin and various systems, leads to organ dysfunction, disability and harms the physical and mental health of patients.¹ Currently, prolonged use of commonly used drugs for scleroderma, such as penicillamine and colchicine, may lead to kidney damage, bone marrow suppression, muscle and peripheral neuropathy. Extensive clinical practice has demonstrated the safety and efficacy of Traditional Chinese Medicine in treating scleroderma.² However, there remains a scarcity of standardized and comprehensive studies elucidating its therapeutic mechanisms.

Fibrosis is the key pathological change in scleroderma, and research indicates a close association between fibrosis and the autophagy process.³ Autophagy, a vital cellular homeostasis mechanism involving the degradation of damaged organelles and proteins via lysosomes, exhibits relevance in various fibrosis-related diseases. Studies suggest that aberrant autophagy contributes significantly to fibrosis pathogenesis, where defects in autophagy within fibroblasts result in excessive extracellular matrix (ECM) and hinder collagen degradation.⁴ Moreover, investigations highlight the potential of autophagy as a promising intervention target in pulmonary fibrosis. Modulating fibroblast apoptosis through autophagy regulation shows promise in alleviating fibrosis progression.⁵

The protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway is a key regulator of autophagy, susceptible to inhibition by reactive oxygen species (ROS).^6,7 Recent researches suggest that phosphorylated Akt transmits signals to activate mTOR, which is the critical factor in autophagosome formation. Recent studies highlight the role of phosphorylated Akt in transmitting signals to activate mTOR, a pivotal factor governing autophagosome formation. Downstream of the Akt-mTOR pathway, a range of autophagy-related factors exists, including B-cell lymphoma-2-interacting myosin-like coiled-coil protein 1 (Beclin-1), which was identified in 1998 and subsequently named Beclin-1. This protein plays a positive regulatory role in autophagy by engaging with co-factors, forming the Beclin-1 interactome.⁸ This interactome induces microtubule-associated protein A/B- light chain 3 (LC3), locating on the autophagosome membrane, to regulate the formation and maturation of autophagosomes.⁹

Huoxue Chubi decoction (活血除痹汤, HXCB), formulated by Prof. DUAN Xingwu, has been a staple at Dongzhimen Hospital Beijing University of Chinese Medicine for several years, demonstrating promising clinical efficacy in treating scleroderma.^10,11 This study aims to delve into the mechanisms underlying the impact of HXCB on skin fibrosis in a scleroderma mouse model. Specifically, we seek to elucidate how HXCB may modulate ROS-mediated autophagy via the Akt-mTOR pathway.

2. MATERIALS AND METHODS

2.1. Preparation of HXCB decoction

HXCB decoction was prepared containing 10 g Danggui (Radix Angelicae Sinensis), 10 g Baishao (Radix Paeoniae Alba), 15 g Dasnhen (Radix Salviae Miltiorrhizae), 10 g Chuanxiong (Rhizoma Chuanxiong), 30 g Jixueteng (Caulis Spatholobi), 10 g Guizhi (Ramulus Cinnamomi), 10 g Duzhong (Cortex Eucommiae), 15 g Huangqi (Radix Astragali Mongolici), 15 g Qinjiao (Radix Gentianae Macrophyllae), 10 g Fangfeng (Radix Saposhnikoviae), 15 g Qiyeyizhihuagen (Rhizoma Paridis) and 10 g Xixiancao (Herba Siegesbeckiae Orientalis). Formula granules of HXCB decoction were purchased from Kang Ren-tang Pharmaceutical Co., Ltd. (Beijing, China). The final concentration of 0.46 g/mL HXCB decoction was prepared and stored in a sterile glass bottle at 4 ℃ for later use.

2.2. Animals and groups

There are 42 male Babl/c mice (6-8 weeks old, weighing 18-20 g) of specific pathogen free grade procured from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). They were then randomly allocated into 6 groups by random number table method: the normal group, scleroderma model group [bleomycin (BLM) group], colchicine group, HXCB high dose (HXCB-H) group, HXCB medium dose (HXCB-M) group and HXCB low dose (HXCB-L) group, with each group comprising 7 mice. The mice were housed in plastic cages (four mice per cage) at room temperature (21 ± 3) ℃ and 25%-35% humidity, following a 12∶12 hour light and dark cycle.

2.3. Mouse scleroderma model and drugs dosage

Following 3 d of acclimation to their environment, the mice underwent a hair removal procedure on a 3 cm × 3 cm area on their backs, ensuring the absence of any allergic or inflammatory reactions in the treated regions. Starting from the 4th day until the 32nd day, all mice, excluding those in the normal group, received subcutaneous 0.1 mL injections of 400 μg/mL BLM at the center of the depilated area to induce the scleroderma mouse model. Meanwhile, mice in the normal group were injected with 0.1 mL of sterile water at the same location.

Upon successful establishment of the BLM-induced model, each mouse received 0.2 mL of the intervention drug for 28 consecutive days via oral gavage. The normal group and model group received daily gavages of sterile water, the colchicine group received daily gavages of colchicine at a dosage of 0.13 mg·kg^-1·d^-1, and the remaining groups were administered HXCB at high, medium, and low doses, with concentrations of 4.6, 2.3, and 1.15 g·kg^-1·d^-1, respectively. On the 60th day, all mice were euthanized, and skin tissues from the injection sites were collected for further analysis.

2.4. Weight measurement of mice

The weight of the mice in each group was weighed at a fixed time before modelling, after modelling and at the end of treatment. The weight of the mice on the third day was used as the base weight.

2.5. Hematoxylin and eosin (HE) staining

A portion of the skin tissues were fixed in 10% formalin, embedded in paraffin and cut into 5 μm thick sections. These tissue samples were stained with HE and examined under an Olympus DX41 microscope (Olympus Corporation, Tokyo, Japan) at two randomly selected fields under 100 × magnification. The average epidermal and dermal thickness was quantified at each field utilizing the Image Pro Plus (version 6.0.0.260, Media Cybernetics, Rockville, MA, USA) image analysis system.

2.6. Masson-trichrome staining

The Masson-trichrome staining method is a well-established technique for highlighting collagen and muscle fibers selectively. It capitalizes on the varied molecular sizes, rendering muscle fibers in red and collagen fibers in blue. Each 5 μm thick section was examined at six randomly chosen 100 × fields using a binocular Olympus DX41 microscope (Olympus Corporation, Tokyo, Japan). Image Pro Plus (version 6.0.0.260, Media Cybernetics, Rockville, MA, USA) image analysis system was used to measure integrated optical density (IOD) and area of the positive (blue area) in each field, and to calculate average optical density of the blue area (positive IOD/Area) representing collagen content per unit area.

2.7. LC3 immumohistochemical staining

After deparaffinization and rehydration, 5 μm thick sections were repaired in 0.01% sodium citrate buffer and blocked with 3% peroxide-methanol at room temperature to eliminate endogenous peroxidase activity. These sections were then subjected to overnight incubation at 4 ℃ with rabbit monoclonal anti-(microtubule-associated protein A/B-light chain 3) LC3AB antibody (1∶1000, Cat#12741S, CST, Danvers, MA, USA), followed by incubation with goat anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP) (Cat# PV-6001, ZSGB, Beijing, China) at 37 ℃ for 20 min. The sections were then subjected to coloration with 3,3-diaminobenzidine (DAB) at room temperature in the absence of light for 10 min.

2.8. ROS detection

The fresh skin tissues collected from each mouse were added to 1 mL of phosphate-buffered saline (PBS), subsequently crushed in an ice bath, and then subjected to centrifugation at 4 ℃. The resulting supernatant from centrifugation was collected, and the protein content was determined using a bicinchoninic acid (BCA) protein assay (Cat#02912E, cwbiotech, Beijing, China). Additionally, the ROS levels were quantified utilizing a ROS Assay Kit (Cat# E004-1-1, NJJC, Nanjing, China). The test results were presented as IOD/mg protein.

2.9. Western blotting

The protein extracted from fresh skin tissues was resuspended in 0.5 mL of radio immunoprecipitation assay lysis buffer (Cat#P0013B, BiYunTian, Shanghai, China). For phosphorylated protein detection, Protease/ Phosphatase Inhibitor Cocktail (Cat#5872, CST, Danvers, MA, USA) was added to the RIPA lysis buffer. Following centrifugation at 4 ℃ for 5 min at 10 000 rpm, the sample protein levels were quantified using a BCA protein assay (Cat#02912E, cwbiotech, Beijing, China). Subsequently, all samples underwent electrophoresis on sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) and were transferred onto polyvinylidene difluoride (PVDF) membranes.

The PVDF membranes were then blocked with 5% nonfat milk at room temperature for 1 h. Each membrane was individually incubated overnight at 4 ℃ with the respective primary antibodies as follows: rabbit polyclonal anti-Akt (phospho T308) (1∶1000, Cat# ab38449, Abcam, Cambridge, UK), rabbit polyclonal anti-pan-Akt (1∶500, Cat#ab8805, Abcam, Cambridge, UK), rabbit monoclonal anti-mTOR (phospho S2448) (1∶1000, Cat#ab109268, Abcam, Cambridge, UK), rabbit monoclonal anti-mTOR (1∶1000, Cat#ab32028, Abcam, Cambridge, UK), rabbit monoclonal anti-LC3AB antibody (1∶1000, Cat#12741S, CST, Danvers, MA, USA), rabbit monoclonal anti-Beclin-1 (1∶1000, Cat#ab210498, Abcam, Cambridge, UK), and rabbit polyclonal anti-GAPDH (1∶2000, Cat#10494-1-AP, proteintech, Wuhan, China).

After three washes with Tris-Buffered Saline Tween-20 (TBST), the PVDF membranes were exposed to goat anti-rabbit IgG (H + L)-HRP (Jackson, West Grove, PA, USA) secondary antibody at room temperature for 1 h. Post additional washes, membrane detection occurred via the chemiluminescence method (ECL, Santa Cruz, CA, USA), and imaging was performed using gel imaging system (ver.4.00., Bio-Rad, Hercules, CA, USA). GAPDH served as the loading control.

2.10. Qualitative reverse transcription polymerase chain reaction (qRT-PCR)

Total tissue RNA was extracted using a RNAprep Pure Tissue Kit (Qiagen, Germantown, MD, USA) and reversed transcribed using a PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan). Quantitative PCR was performed using SYBR® Premix Ex Taq™ Ⅱ(TaKaRa, Shiga, Japan) following the manufacturers’ protocols. PCR was performed with the following conditions: pre-degeneration at 95 ℃ for 3 min, denaturation at 95 ℃ for 15 s, annealing at 60 ℃ for 1 min, for a total of 40 cycles. The relative expression of target genes was calculated using the 2^-ΔΔCt method. β-actin was used as the internal control for qRT-PCR. The PCR primer sequences were designed according to their gene sequences reported in GenBank. LC3: forward 5’-CACAGCATGGTGAGTGTGTC-3’, reverse 5’-TCAGAAGCCGAAGGTTTCCT-3’; Beclin-1: forward: 5′-AACCTCAGCCGAAGACTGAA-3′, reverse: 5′-CAGTGACGTTGAGCTGAGTG-3′, β-actin: forward: 5′-TGGACTTCGAGCAAGAGATG-3′; reverse: 5′- GAAGGAAGGCTGGAAGAGTG-3′.

2.11. Statistics analysis

Data were shown as mean ± standard deviation ($\overline{x}\pm s$). Statistics were calculated using the GraphPad Prism 7.0 software (GraphPad Software, La Jolla, CA, USA) and SPSS 17.0 (IBM Corp., Armonk, New York, USA).

The single factor analysis of variance of completely randomized design was used for parameter comparison between groups. Correlation analyses were conducted using the Spearman correlation coefficient test. Significance levels were denoted as P < 0.05, P < 0.01, and P < 0.001.

3. RESULTS

3.1. HXCB inhibited weight loss in scleroderma mice

In the control group, mice exhibited a consistent and steady increase in weight. However, in comparison to the control group, scleroderma-afflicted mice across all groups experienced a decline in weight by day 31 (P < 0.001). Post-treatment, mice in each drug intervention group demonstrated a gradual increase in weight compared to the model group by day 60 (P < 0.01), with notably significant improvements observed in the colchicine group and HXCB-H group (P < 0.001).

3.2. HXCB reduced skin thickness in scleroderma mice

HE staining revealed a progressive increase in epidermal and dermal thickness in scleroderma model mice (P < 0.001), concomitant with heightened and thickened collagen fibers, structural disturbances, deep staining, dermal infiltration by inflammatory cells, atrophied hair follicles, and diminished fat layers compared to the control group. Following treatment, mice in each drug intervention group exhibited a reduction in epidermal and dermal thickness, notably observed in the colchicine group and HXCB-H group (P < 0.05). Additionally, other pathological changes showed improvements (Table 1 and Figure 1).

Table 1.

Comparison of skin thickness, ROS and collagen levels in each group ($\overline{x}\pm s$)

Group	n	Skin thickness (μm)	ROS (IOD/mg)	Collagen level (IOD/μm2)
Control	7	333.91±29.44a	1616.40±295.02 a	0.26±0.05c
BLM	7	544.36±77.19	9311.22±1790.95	0.43±0.09
Colchicine	7	436.81±50.84b	3495.15±1976.69c	0.28±0.03c
HXCB-H	7	443.82±42.65b	4681.03±1020.88c	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

Notes: 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, HXCB-H: high-dose Huoxue Chubi decoction; HXCB-M: middle-dose Huoxue Chubi decoction; HXCB-L: low-dose Huoxue Chubi decoction; ROS: reactive oxygen species; IOD: integrated optical density. The single factor analysis of variance of completely randomized design was used for parameter comparison between groups. ^aP < 0.001, ^bP < 0.05, ^cP < 0.01, vs BLM group.

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.

3.3. HXCB reduced dermal collagen content in scleroderma mice

Masson-trichrome stained sections revealed a progressive increase in dermal positive IOD/Area in scleroderma model mice compared to the control group (P < 0.01). Post-treatment, the dermal positive IOD/Area exhibited a decrease in each drug intervention group, particularly notable in the colchicine group and HXCB-H group (P < 0.05) (Table 1 and Figure 2).

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.

3.4. HXCB reduced cutaneous ROS levels in scleroderma mice

In comparison to the control group, scleroderma model mice exhibited a significant up-regulation in cutaneous ROS expression (P < 0.001). Post-treatment, the expression of cutaneous ROS displayed a gradual decrease in both the colchicine group and the HXCB-H group compared to the scleroderma model group (P < 0.01 and P < 0.001) (Table 1).

3.5. HXCB controlled Akt-mTOR pathway activation in scleroderma mice

Phospho-Akt (p-Akt)/Akt and Phospho-mTOR (p-mTOR)/mTOR ratios are indicative of Akt-mTOR pathway activation levels. In contrast to the control group, the scleroderma model group exhibited a significant increase in the expression of p-Akt/Akt and p-mTOR/ mTOR (P < 0.001). Following treatment, all drug intervention groups showed significant down-regulation in the expression of p-Akt/Akt and p-mTOR/mTOR (P < 0.001). Notably, the expression levels of p-Akt/Akt and p-mTOR/mTOR in each HXBC decoction group demonstrated a dose-dependent response, escalating with the intervention concentration (Figures 3A and 3B).

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.

Simultaneously, mRNA expression of Akt and mTOR significantly rose in the scleroderma model group compared to the control group (P < 0.001). However, post-treatment, mRNA expression of Akt and mTOR substantially decreased in all drug intervention groups (P < 0.001). Furthermore, the mRNA expression of Akt and mTOR in each HXBC decoction group exhibited a dose-dependent pattern (Table 2).

Table 2.

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

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

Notes: qRT-PCR was performed to detect mRNA expression of Akt, mTOR, Beclin-1 and LC3 in each group of scleroderma mice. 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. mRNA: messenger ribonucleic acid; mTOR: mammalian target of rapamycin; LC3: microtubule-associated protein A/B-light chain 3; Beclin-1: B-celllymphoma-2-interacting myosin-like coiled-coil protein 1; BLM: bleomycin; HXCB-H: high-dose Huoxue Chubi decoction; HXCB-M: middle-dose Huoxue Chubi decoction; HXCB-L: low-dose Huoxue Chubi decoction; qRT-PCR: qualitative reverse transcription polymerase chain reaction. The single factor analysis of variance of completely randomized design was used for parameter comparison between groups, ^aP < 0.001 vs BLM group.

3.6. HXCB decoction enhanced the protein and mRNA expression of autophagy factor LC3 and Beclin-1 in scleroderma mice

Compared to the control group, both the protein and mRNA expression levels of autophagy factors LC3 and Beclin-1 decreased significantly in the scleroderma model group (P < 0.001). However, following treatment, there was a substantial up-regulation observed in the protein and mRNA expression of LC3 and Beclin-1 across all drug intervention groups (P < 0.001 or P < 0.01). Interestingly, within each HXBC decoction group, the protein and mRNA expression of LC3 and Beclin-1 exhibited a dose-dependent decrease correlated with the intervention concentration (Figures 3C, 3D, and Table 2).

LC3 immunohistochemical staining displayed predominant LC3 positive expression in the epidermis, skin appendages, and fibroblasts. Notably, the control group and drug intervention groups exhibited robustly positive LC3 expression, whereas the scleroderma model group displayed weakly positive LC3 expression (Figure 4).

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.

3.7. Correlation analysis

The statistical analysis revealed significant correlations within the data. The expression of ROS exhibited a positive correlation with skin thickness (r = 0.69, P < 0.001), collagen content (r = 0.52, P < 0.001), and the mRNA expression of Akt (r = 0.38, P < 0.05). Conversely, the expression of p-Akt/Akt and p-mTOR/ mTOR showed negative correlations with the protein expression of LC3 (r = －0.59, P < 0.05 and r = －0.82, P < 0.001) and Beclin-1 (r = －0.87, P < 0.001 and r = －0.78, P < 0.001). Similarly, the mRNA expression of Akt and mTOR exhibited negative correlations with the mRNA expression of LC3 (r = －0.59, P < 0.001 and r = －0.71, P < 0.001) and Beclin-1 (r = －0.85, P < 0.001 and r = －0.88, P < 0.001).

4. DISCUSSION

Scleroderma, called "Pi Bi" in ancient Chinese medical books, is caused mainly by the deficiency of Yang Qi and the inability of defensive Qi to protect against wind-cold-dampness evil. This leads to obstruction of channels and collaterals, stagnation of Qi and blood and disharmony between nutritive Qi and defensive Qi.¹² "Qi deficiency and blood stasis" is the core pathogenesis throughout the development of scleroderma.¹³ HXCB decoction, developed by Prof. DUAN Xingwu has demonstrated notable efficacy in treating scleroderma. Within this decoction, Danggui (Radix Angelicae Sinensis) and Huangqi (Radix Astragali Mongolici) act as sovereign drugs, fortifying Qi, nourishing blood, and enhancing blood circulation. Jixueteng (Caulis Spatholobi), Chuanxiong (Rhizoma Chuanxiong), and Danshen (Radix Salviae Miltiorrhizae) serve as minister drugs, warming meridians, unblocking collaterals, and alleviating pain. Adjuvant drugs like Qinjiao (Radix Gentianae Macrophyllae), Fangfeng (Radix Saposhnikoviae), Qiyeyizhihuagen (Rhizoma Paridis), Duzhong (Cortex Eucommiae), Xixiancao (Herba Siegesbeckiae Orientalis), Guizhi (Ramulus Cinnamomi), and Baishao (Radix Paeoniae Alba) collectively disperse wind, eliminate "Bi," dissolve masses, clear toxins, and harmonize Ying and Wei energies. This formula functions to invigorate Qi, promote blood circulation, facilitate the free flow of Yang Qi, and eliminate "Bi."

The excessive deposition of collagen in skin tissues and internal organs leads to fibrosis, significant pathological mechanism in scleroderma.¹⁴ Autophagy, an evolutionarily conserved cellular process, degrades "endogenous stasis" like excessive collagen deposition via lysosomes, thereby mitigating organ and tissue fibrosis and achieving the objective of "eliminating blood stasis and generating new tissues.".^15,16 The Akt-mTOR signaling pathway plays a crucial role in regulating autophagy. mTOR, a pivotal initiator of autophagy, can be activated directly or indirectly by phosphorylated Akt. Akt-mTOR is an important signaling pathway for autophagy regulation.¹⁷ Consequently, mTOR negatively regulates Beclin-1, a vital factor in autophagy formation, subsequently influencing LC3, a key downstream molecule post-autophagy, thus inhibiting autophagy occurrence.^{18,⇓- 20}

In this study, mice injected with bleomycin displayed a significant elevation in ROS levels, increased skin thickness, and heightened collagen levels consistent with the pathological changes observed in scleroderma mice. Concurrently, there was a significant increase in the protein and mRNA expression levels of Akt-mTOR pathway-related factors alongside a significant decrease in the protein and mRNA expression levels of autophagy related factors Beclin-1 and LC3. These findings strongly indicate an activated Akt-mTOR pathway and suppressed autophagy in the skin of scleroderma mice. Correlation analyses unveiled positive associations between ROS and skin thickness, collagen content, and Akt mRNA expression, while negative correlations emerged between Akt-mTOR pathway-related factors and Beclin-1/LC3 expression at both protein and mRNA levels. These results suggested that that oxidative stress can prompt fibroblasts to enhance collagen synthesis, potentially linked to ROS-triggered activation of the Akt-mTOR autophagy pathway, thus impeding collagen degradation and accelerating its deposition. Subsequent treatment with HXCB resulted in decreased ROS levels, diminished skin collagen hyperplasia, reduced Akt-mTOR pathway activity in scleroderma mice, and enhanced levels of autophagy-related factors Beclin-1 and LC3. This suggests that HXCB might foster autophagy through the Akt-mTOR pathway, augmenting collagen degradation by mitigating tissue oxidative stress response, consequently curtailing skin collagen synthesis.

In summary, HXCB showcases its potential in regulating autophagy by fortifying Qi and enhancing blood circulation, thereby mitigating blood stasis and facilitating tissue regeneration in scleroderma treatment. Nonetheless, the intricate molecular biological mechanisms underlying how HXCB mitigates autophagy necessitate further exploration, a focus we aim to delve into through forthcoming cell experiments.

Funding Statement

Supported by Natural Science Foundation-funded Project: Exploration the Mechanism of Yiqi Huoxue Therapy in Treating Scleroderma Fibrosis based on Phosphatidylinositol 3-Kinase (PI3K)-Protein Kinase B (Akt)-Mammalian Target of Rapamycin (mTOR) Signal Pathway about Autophagy (No. 81804106), and the 2023 Science and Technology Innovation Project Dongzhimen Hospital Beijing University of Chinese Medicine: Exploration the Mechanism of Radix Salviae Miltiorrhizae Components in Treating Scleroderma Fibrosis Based on PI3K-Akt-mTOR Signal Pathway about Autophagy (No. DZMKJCX-2023-009)