Amelioration of Oxidative Stress in Rats with Chronic Obstructive Pulmonary Disease through Shenqi Huatan Decoction Activation of Peroxisome Proliferator-Activated Receptor Gamma-Mediated Activated Protein Kinase/Forkhead Transcription Factor O3a Signaling Pathway

Background Chronic obstructive pulmonary disease (COPD) is a common respiratory disease. Currently, no specific treatment strategy has been established; therefore, finding new treatment methods is essential. Clinically, Shenqi Huatan Decoction (SQHT) is a traditional Chinese medicinal formula for COPD treatment; however, its mechanism of action in treatment needs to be clarified. Methods The COPD rat model was replicated by cigarette smoking and tracheal injection using the LPS method. The control group and the SQHT groups were treated with dexamethasone and SQHT by gavage, respectively. After treatment, superoxide dismutase (SOD) serum levels, total antioxidant capacity (TAOC), lipid peroxidation, and malondialdehyde (MDA) were detected by enzyme-linked immunosorbent assay (ELISA). Activated protein kinase alpha (AMPK-α), forkhead transcription factor O3a (FOXO3a), manganese SOD (MnSOD), and peroxisome proliferator-activated receptor gamma (PPARγ) were detected using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and Western blot. Microribonucleic acid and protein expression levels were measured, and pathological changes in lung tissue were observed using hematoxylin and eosin staining. Results The pathological findings suggested that SQHT substantially affects COPD treatment by enhancing alveolar fusion and reducing emphysema. ELISA results showed that SQHT could lower the blood levels of MDA and lipid peroxide and raise SOD and TAOC levels, suggesting that it could lessen oxidative stress. In the lung tissue of rats with COPD, large doses of SQHT intervention dramatically increased AMPK protein expression, AMPK-α, FOXO3a, MnSOD, and PPARγ, indicating that SQHT may reduce oxidative stress by activating the PPARγ-mediated AMPK/FOXO3a signaling pathway. Similar results were obtained using RT-qPCR. Conclusion SQHT is effective for COPD treatment. The mechanism of action may be related to the activation of the PPARγ-mediated AMPK/FOXO3a signaling pathway to improve oxidative stress in lung tissue.


Introduction
Chronic obstructive pulmonary disease (COPD) accounts for a high proportion of cases of respiratory diseases, with a high incidence of morbidity and mortality.It is also associated with a high risk of cardiovascular disease, which threatens patients' quality of life and health [1].Te high morbidity, disability, and mortality rates associated with COPD make it a public health issue that must be addressed.Oxidative stress plays a crucial role in the development of COPD [2].Smoking is a signifcant risk factor for COPD.Tobacco smoke contains high levels of oxygen radicals and reactive oxygen species (ROS), which cause airway infammation, leading to membrane lipid peroxidation and structural and functional alterations in various biomolecules, resulting in an imbalance between oxidation and antioxidation [3].Regulation of the intracellular oxidation/ antioxidation balance is essential for COPD prevention and treatment [4].Under physiological conditions, the body's oxidants and antioxidants exist in a dynamic balance; oxidative stress occurs when an imbalance leads to cell death and tissue damage.Owing to their unique tissue structure, rich blood supply, and large respiratory membrane area, the lungs are highly susceptible to damage by endogenous oxidants such as ROS, reactive nitrogen species, and lipid peroxidation products.
Traditional Chinese medicine is a rich theoretical system for treating COPD.Tis system alleviates patients' clinical symptoms and efectively prevents the acute onset of COPD.Te search for corresponding target interventions from traditional Chinese medicine for COPD is of great practical signifcance.Traditional Chinese medicine plays an irreplaceable role in prevention and treatment due to its abundant resources, low economic cost, precise therapeutic efect, seeking the root cause of the disease, and considering both specimens.Te traditional Chinese medicine, Shenqi Huatan Decoction (SQHT), is a combined formula of Shenqi Chongcao capsules and phlegm lowering capsules, consisting of American ginseng, Panax ginseng, Cordyceps, Perillae, white mustard seed, Bai Qian, Golden Boiling Herb, and roasted Ephedra.It has long been used for treating COPD by Professor Han Mingxiang, a master of Chinese medicine, and has defnite results.Previous studies have shown that it improves airway remodeling and reduces airway infation by invading transforming growth factor beta-simulated signaling paths in active sites of modeling and clustering [5].Tis study explored the potential mechanism of action of SQHT in reducing oxidative stress in rats with COPD by activating the peroxisome proliferatoractivated receptor gamma (PPARc)-mediated activated protein kinase (AMPK)/forkhead transcription factor O3a (FOXO3a) signaling pathway at the cellular level.

Animals.
In total, 60 healthy male Sprague-Dawley rats with a specifc pathogen-free rating, aged 4-6 weeks, and weighing an average of 120 ± 10 g were bought from Beijing Huafukang Laboratory Animal Co. (China).Using the random number table method, the 60 rats were randomly divided into six groups of 10 rats each.Te groups are blank, model, dexamethasone, and SQHT high-, medium-, and low-dose.In the blank group, physiological saline was injected without medication and gavage.No medication was administered after modeling in the model group, and physiological saline was administered by gavage.In the dexamethasone group, dexamethasone tablets were administered after modeling.In the low-dose SQHT group, after modeling, the low-dose Shenqi Huatan formula was administered orally.In the middle-dose SQHT group, after modeling, a middle dose of the Shenqi Huatan formula was administered by gavage.In the high-dose SQHT group, after modeling, the high-dose Shenqi Huatan formula was administered orally.Te experimental design is shown in Figure 1.

Induction of COPD in Rats. Te Experimental Animal
Ethics Committee of the Institute of Shanxi Traditional Chinese Medicine in Taiyuan, China, approved this study (no.SZYLY2022KY-0514).All methods were performed in accordance with the relevant regulations and animal research: Reporting of In Vivo Experiment Guidelines.Te animals were allowed to adapt to the environment for one week before starting the experiment.
Te COPD rat model was replicated by cigarette smoking and tracheal injection using the LPS method [6].LPS was injected into the trachea of rats on days 1 and 14 at a concentration of 0.2 ml/rat in the blank group.Rats in the blank group were administered a similar volume of 0.9% saline.On days 2-13 and 15-40, rats passively smoked 10 cigarettes per day for 40 min.

Drug Administration.
Dosing was started at the end of the modeling and administered once daily for 14 days.
(i) Te blank and model groups were administered 0.9% sodium chloride solution by oral gavage.
2 Canadian Respiratory Journal (ii) Te dexamethasone group was gavaged with a dexamethasone suspension using an equivalent dose conversion formula of 0.75 mg/60 kg × 7.5 � 0.09 mg/kg daily for rats.(iii) In the treatment group, rats were administered SQHT by gavage.Te daily doses of the drug in the high-, medium-, and low-dose groups were 15, 7.5, and 3.8 times the clinical dose, respectively.Te daily drug doses were 8.6 g/kg, 4.3 g/kg, and 2.2 g/kg for the high-, medium-, and low-dose groups, respectively.

Hematoxylin and Eosin Staining to Observe Histopathological Changes in Rat
Lungs.After the rats were anesthetized with 10% pentobarbital sodium, the skin of the chest and abdomen were cleaned with 75% ethanol, and the muscles from the abdomen to the neck were cut under sterile conditions.Blood was withdrawn from the inferior vena cava and both lungs were removed.Te right lung was immediately frozen in an ultralow-temperature refrigerator at −80 °C for testing.Te left lung was fxed in 4% paraformaldehyde solution, embedded in parafn, sectioned, and stained with H&E stain.Te structural changes in the alveoli and airway walls were observed under a microscope.

SOD, TAOC, LPO, and MDA Levels in Rat Serum Were
Measured by ELISA.After blood was collected from the inferior vena cava, it was centrifuged using a high-speed centrifuge at 1500 rpm for 20 min, and the supernatant was collected and tested in strict accordance with the ELISA kit instructions.
Te mRNA expression of AMPK-α, FOXO3a, MnSOD, and PPARc was detected using real-time fuorescence quantitative PCR Te total ribonucleic acid (RNA) from lung tissues was extracted by the TRIzol method, incubated at 42 °C for 15 min and 85 °C for 5 min, and reverse transcribed to cDNA.PCR amplifcation was performed using this template.Te reaction conditions were as follows: step 1, predenaturation at 95 °C for 15 min, and step 2, 95 °C for 20 s, 55 °C for 30 s, and 72 °C for 30 s, 40 cycles.Primers were designed and synthesized by Sangon Biotech Co., Ltd.(Shanghai, China), and the specifc sequences are listed in Table 1.Te relative quantifcation of each indicator microRNA was carried out using the 2 −△△CT method with β--actin as the internal reference gene.

Protein Expression of AMPK-α, FOXO3a, p-FOXO3a, MnSOD, and PPARc Was Detected by Western Blot.
Lung tissues were lysed in a radioimmunoprecipitation assay bufer.Te lysate was centrifuged at 12000 rpm for 15 min at 4 °C, and the supernatant was collected for further analysis.Te total protein concentration was determined using a Bicinchoninic acid protein assay kit.Following electrophoresis and transfer, the membrane was blocked with 5% nonfat dry milk for 1 h at room temperature.Te primary antibody was diluted as instructed, and the membrane was incubated with primary antibodies overnight at 4 °C.After washing with Tris-bufered saline with 0.1% Tween 20 detergent, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature.Te polyvinylidene fuoride membranes were then incubated with chemiluminescent Canadian Respiratory Journal reagents to visualize the bands.Te relative expression of the target protein was expressed as the ratio of the target protein's gray value to the internal reference, using GAPDH as the internal reference.ImageJ software was used to detect the gray values of the western blot bands.

Statistical Analysis.
Statistical analysis was performed using SPSS 26.0 and GraphPad Prism 8.0.Te experimental data are all measures expressed as mean ± standard deviation.If the distribution was normal, one-way analysis of variance was chosen as the statistical method, and if the variance was the same, Tukey's post hoc analysis was chosen for multiple comparisons between the two groups.P < 0.05 indicated statistically signifcant diferences, whereas P < 0.01 indicated highly statistically signifcant diferences.

Histopathological Sections of the Lung.
Te alveolar septum between adjacent alveoli in the blank group is relatively thin, with clear boundaries, complete alveolar wall structure, no fusion between alveoli, and no obvious abnormalities.In the model group, alveolar consolidation, alveolar fusion, widened septa, development of pulmonary bullae, infltration of a large number of granulocytes and macrophages in the airway and between the alveoli, and scattered distribution of a small number of granulocytes, macrophages, and foam cells in the surrounding alveoli can be seen.At the same time, bleeding was present in the small bronchi.Both the control group and the low-dose, mediumdose, and high-dose SQHT groups showed varying degrees of alveolar enlargement, partial alveolar fusion, uneven infltration of infammatory cells around the tracheal wall, partial shedding of tracheal mucosal epithelium, and varying degrees of narrowing of the lumen.After treatment, the alveolar status in the SQHT groups and the dexamethasone group improved compared to the model group, with reduced infltration of infammatory cells and no bronchial bleeding observed.Te SQHT high-dose group and the dexamethasone group had the best state (Figure 2).Based on the INHAND Proposal (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) [7], three samples from each group were selected for pathological scoring, with the specifc results listed in Table 2.

SQHT Can Increase the Content of SOD and TAOC and
Reduce the Content of LPO and MDA.Compared with the blank group, serum SOD and TAOC in the rats of the model group were signifcantly reduced, and MDA and LPO were signifcantly increased (P < 0.01).Compared with the model group, MDA and LPO were signifcantly lower, and SOD and TAOC were signifcantly higher in each treatment group (P < 0.01), with the most signifcant diference in the highdose group (Figure 3).

SQHT Signifcantly Increased the mRNA Expression of AMPK-α, FOXO3a
, MnSOD, and PPARc.Te lung tissue of rats in the model group had signifcantly lower mRNA expression levels of AMPK-α, FOXO3a, MnSOD, and PPARc than the blank group (P < 0.01).In comparison with the model group, the control and SQHT medium-and highdose groups had signifcantly increased mRNA expression of AMPK-α and FOXO3a (P < 0.01), the high-dose group only had signifcantly increased mRNA expression of MnSOD (P < 0.01), and the control and high-dose groups had signifcantly increased mRNA expression of PPARc (P < 0.05 and P < 0.01, respectively) (Figure 4).

SQHT Signifcantly Increased the Protein Expression
Levels of AMPK-α, FOXO3a, p-FOXO3a, MnSOD, and PPARc.Compared with the blank group, the protein expression levels of AMPK-α, FOXO3a, p-FOXO3a, MnSOD, and PPARc were signifcantly lower in the lung tissues of rats in the model group (P < 0.01).Compared with the model group, the abovementioned indices were signifcantly higher in the control and SQHT medium-and high-dose groups (P < 0.01), with the most signifcant diference in the control group (Figure 5).

Discussion
Oxidative stress plays a vital role in COPD pathogenesis and is mainly caused by the accumulation of endogenous and exogenous oxidants.Te primary sources of exogenous ROS are cigarette smoke and airborne particulate matter [8].High levels of free radicals and other oxidants in cigarette smoke dissolve in the fuid that lines the airway epithelium upon entry into the airways, accumulate in the lungs, irritate the    indicator of oxidative stress in COPD, the TAOC, might represent the antioxidant status [11].SOD is an endogenous antioxidant and metalloactive enzyme that scavenges oxygen radicals.It provides insights into the body's free radical metabolism and is crucial for maintaining a proper balance between oxidative stress and antioxidant defenses [12].
Studies have shown that airway infammation in patients with COPD can lead to impaired oxygen exchange, resulting in ischemia and hypoxia in the lungs and reduced SOD activity [13].Oxidants readily oxidize lipids and lipid peroxidation is a major consequence of oxidative stress.LPO damages vascular endothelial cells, alters biological 6 Canadian Respiratory Journal membrane structure, increases the number of leukocytes, promotes platelet chemotaxis and aggregation, and promotes free radicals production, thereby inhibiting the body's immune response [14].Enhanced peroxidation in the body increases the level of LPO, which reduces its ability to scavenge oxygen radicals, thereby reducing SOD activity and causing abnormalities in cell structure [15].MDA is one of the end products of peroxidation reactions, which can indirectly represent the severity of cellular attack by free radicals [16], and is a marker of lipid peroxidation.It can cross-link macromolecules in organisms, including proteins, nucleic acids, and lipids, leading to bioflm degradation and cell death due to reactions between ROS and unsaturated fatty acids.Te results of this study showed that SOD and TAOC levels were signifcantly lower and MDA and LPO levels were signifcantly higher in the serum of COPD rats.Following therapy, the levels of MDA and LPO were signifcantly lower and those of SOD and TAOC were signifcantly higher.Te therapeutic efects of SQHT and dexamethasone were demonstrated by improved oxidative stress and enhanced antioxidant capacity in rats with COPD.
PPARc belongs to the superfamily of ligand-activated transcription factors, which recognize lipid oxidation products such as oxidized phospholipids and nitroolefns to sense oxidative stress and inhibit oxidative stress by regulating oxidase activity [17].By activating several pathways to limit the body's exposure to free radicals and by competitively suppressing the infammatory mediators produced by associated infammatory signaling pathways, PPAR is a crucial transcription factor in the body's infammatory response and oxidative stress [18].
Te adenylate-AMPK/FOXO3a pathway plays a vital role in reducing ROS accumulation of reactive oxygen species and combating oxidative and infammatory responses [19].AMPK, a crucial cellular protein kinase present in cells, regulates energy and metabolism in the body.Tis process requires intricate signaling pathways and regulatory processes.It has been found that ROS produced by mitochondria can act as a signaling molecule to activate AMPK [20], which suggests that AMPK is redox-sensitive.AMPK activation reduces ROS accumulation and protects fbroblasts against oxidative stress damage [21].FOXO3a is a member of the transcription factor family that is important for cell proliferation, apoptosis, and oxidative stress [22].As a direct downstream target of AMPK, FOXO3a is nucleated when AMPK is activated, which increases the activity of ROS-detoxifying enzymes, reduces ROS-induced stress, and aids in cell survival.Catalase (Cat) and MnSOD are direct targets of FoxO3a.Terefore, increased levels of Cat and MnSOD following FoxO3a activation could efectively manage ROS levels and reduce ROS-induced stress [23].MnSOD, a form of SOD, is an essential endogenous antioxidant protein and a member of the metalloantioxidant enzyme family.AMPK activation has also been reported to upregulate MnSOD levels [24].PPARc plays an essential biological function in cell diferentiation and metabolic regulation.By regulating downstream forkhead proteins, PPARc can phosphorylate AMPK, thereby activating the AMPK/FOXO3a signaling pathway and exerting antioxidant efects [25].Wang [26] found that PPARc signifcantly increased AMPK activity and protected airway epithelial cells from infammatory damage.
In accordance with the results of our investigation, PPARc markedly boosted AMPK activation and shielded airway epithelial cells from infammatory damage.In this experiment, the protein expression levels of AMPK-α, FOXO3a, p-FOXO3a, MnSOD, and PPARc in the lung tissues of the COPD model group were signifcantly lower than those of the blank group.In contrast, they were signifcantly higher compared to the model group and in the control group and the SQHT medium-dose and high-dose groups.Terefore, it can be concluded that the activation of PPARc can enhance the expression of AMPK, which can induce the nucleation of FOXO3a and upregulate the phosphorylation level of FOXO3a.FOXO3a binds to the antioxidant gene MnSOD, upregulates the levels of antioxidant enzymes, scavenges excess oxygen radicals, and reduces oxidative damage.

Conclusions
Tis experiment showed that treatment of COPD rats with SQHT improved the histopathological changes in the lungs with signifcant efcacy.Te mechanism may be related to the activation of the AMPK/FOXO3a signaling pathway by PPARc to reduce oxidative stress in the lungs of rats.

Figure 2 :
Figure 2: Efects of Shenqi Huatan Decoction (SQHT) and dexamethasone on histological changes in hematoxylin and eosin (H&E)-stained lung sections of chronic obstructive pulmonary disease (COPD) rats.Te black arrow indicates extensive infltration by granulocytes and macrophages.Te green arrow symbolizes multifocal hemorrhaging.Te blank triangle represents local lymphocyte infltration around blood vessels and trachea.

Figure 3 :
Figure3: Efect of SQHTand DEX on the expression of SOD, TAOC, LPO, and MDA in serum from chronic obstructive pulmonary disease rats.Compared with the blank group, * P < 0.05 and * * P < 0.01; compared with the model group, # P < 0.05 and ## P < 0.01.

Table 1 :
[10]er sequences for target genes.During oxidative stress in COPD, ROS causes airway epithelial cell damage, induces airway smooth muscle cell proliferation, promotes airway wall thickening, and increases the rate of decline in lung function[10].An essential [9]g tissue over time, sustain infammation, and aid in developing COPD[9].Te infammatory response and hypoxia are essential factors in producing endogenous ROS.An increase in either endogenous or exogenous ROS disrupts the oxidative/antioxidative balance, activating the pulmonary and systemic oxidative stress response in patients with COPD.Regulating oxidative stress is highly signifcant in COPD prevention and treatment.

Table 2 :
Pathological scores (lung tissue).Te higher the total score, the more severe the pathological damage, while the lower the score, the less severe the damage.