Correction: Xinfeng Capsule inhibits oxidative stress via regulating the PPARγ /Hmgcs2 signaling pathway in lung tissue of adjuvant arthritis rats

Methods: Liquid chromatography-mass spectrometry (LC-MS) analysis was conducted to determine the chemical composition of XFC. Proteomic and bioinformatic analyses of differentially expressed proteins (DEPs) in rat lung tissue were performed using tandem mass tag labeling. A rat adjuvant arthritis (AA) model was established using Freund's complete adjuvant to observe pathological changes in synovial and lung tissues, as well as alterations in lung function. In addition, a cell model was constructed by inducing lung fibroblasts with transforming growth factor-β1 (TGF-β1) to investigate the effects of XFCcontaining serum on oxidative stress and pulmonary fibrosis through the peroxisome proliferator-activated receptor gamma (PPARγ)/3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) pathway.Rheumatoid arthritis (RA) is a complex autoimmune disease with multifaceted etiology and mechanisms. The global prevalence of RA is estimated to be between 0.5% and 1% (Finckh et al., 2022). As the disease progresses and recurs, many patients develop pathological changes in tissues and organs beyond the joints. Notably, the lungs are frequently affected in this extraarticular manifestation, involving the lung parenchyma, pleura, and blood vessels. This leads to diverse pathological alterations, including interstitial lung lesions, rheumatoid nodules, and pleuritis (Akiyama & Kaneko, 2022). Among these complications, interstitial lung disease (ILD) is the most common form of pulmonary involvement in RA patients (Samhouri et al., 2022). The reported prevalence of ILD in RA patients varies widely across different cohorts, largely due to differences in detection frequency and diagnostic criteria. Earlier studies estimated that secondary ILD occurs in 1.8%-67% of RA patients (Dai, Wang, Yu, & Hu, 2021;S. Huang et al., 2020). This condition not only adversely affects the quality of life of the patients but also imposes a considerable social and economic burden. Unfortunately, the early stages of RAassociated lung injury often lack obvious clinical symptoms and are instead characterized by a gradual decline in lung function. This insidious onset frequently results in underrecognition and delayed intervention. At present, the precise pathogenesis of lung injury secondary to RA remains poorly understood, and therapeutic options are limited. This knowledge gap highlights the urgent need for effective treatments for this debilitating complication.The peroxisome proliferator-activated receptor γ (PPARγ) signaling pathway has emerged as a key regulator of inflammation, oxidative stress, and fibrosis-central pathological processes in lung injury(J. Liu, Hu, Zhu, Shao, & Yuan, 2020). Activation of PPARγ exerts both antiinflammatory and anti-fibrotic effects in various lung disease models(Q. Li et al., 2024). Notably, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), a critical enzyme in ketogenesis, has recently been identified as a downstream target regulated by PPARγ signaling. Dysregulation of the PPARγ/HMGCS2 axis has been implicated in promoting oxidative stress and cellular damage in extrapulmonary tissues (Hazra, Peebles, Sharma, Mao, & Dubinett, 2008). However, its specific role and therapeutic potential in RA-associated lung injury remain largely unexplored, representing both a major knowledge gap and a promising therapeutic target.Traditional Chinese medicine (TCM) has gained significant attention due to its complex bioactive components and diverse therapeutic targets. Among TCM formulations, Xinfeng Capsule (XFC) is a noteworthy compound preparation produced under strict quality control standards (F. Wang, Meng, Chen, Wang, & Sun, 2014). XFC has been registered in China (Anhui Medicine System Z20050062) and granted a national invention patent (ZL201310011369.8).Comprising Astragali Radix (Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge.), Coicis Semen (Coix lacryma-jobi L. var. ma-yuen (Roman.) Stapf), Tripterygium wilfordii Hook. f., and Scolopendra (Scolopendra subspinipes mutilans L. Koch) at a ratio of 20:20:10:1, XFC has shown considerable therapeutic promise (Gao, Wang, & Meng, 2020). In a previous large-scale, multicenter, randomized, doubleblind clinical trial conducted by our team (Jian et al., 2015), XFC demonstrated significant efficacy in alleviating joint symptoms and improving lung function in RA patients (Yue Sun, Liu, & Wan, 2016;Wan et al., 2016;Yali, Jian, Yuan, & Yue, 2015). Furthermore, animal studies showed that XFC improved lung injury in adjuvant arthritis (AA) rats, while cell-based experiments revealed its ability to inhibit lung fibroblast proliferation (Wan et al., 2017). Despite these encouraging findings, the underlying molecular mechanisms-particularly the potential involvement of the PPARγ/HMGCS2 pathway-remain to be clarified.With advances in molecular biology, proteomic analysis has become a powerful tool in biomedical research, offering a broad perspective for exploring the mechanisms of TCM. In this study, we employed an unbiased, tandem mass tag (TMT)-based proteomic approach to identify novel targets of XFC in the lungs of AA rats. We hypothesized that XFC alleviates RAassociated lung injury by modulating the PPARγ/HMGCS2 signaling pathway to reduce oxidative stress. This study was therefore designed to comprehensively characterize the chemical composition of XFC, validate its efficacy in improving lung function and pathology in AA rats, identify differentially expressed proteins (DEPs) in lung tissue, and mechanistically confirm both in vitro and in vivo that its protective effects are mediated through the PPARγ/HMGCS2 signaling axis. These findings provide novel HMGCS2 insights and additional experimental evidence for a deeper understanding of the pharmacological mechanisms of XFC.XFC is a compound formulation rooted in traditional Chinese medicine. The raw materials used in this preparation were procured from Lejialaopu Pharmacy (Hefei, Anhui Province) and authenticated by Professor Li from the First Affiliated Hospital of Anhui University of Chinese Medicine. All herbal components comply with the specifications outlined in the 2020 edition of the Chinese Pharmacopoeia and were manufactured in strict accordance with national regulatory standards. The formulation consists of Astragalus membranaceus (Batch No. 2208015, Inner Mongolia, China), Coix lacryma-jobi L. var (Batch No. YPC2F0012, Fujian, China), Scolopendra subspinipes mutilans L. Koch (Batch No. 2207008, Hubei, China), and Tripterygium wilfordii Hook. f (Batch No. YPT1G0005, Guizhou, China), with a defined dosage ratio of 20:20:10:1. In prior research, our team established a high-performance liquid chromatography (HPLC)-based fingerprint profile, which demonstrated that XFC possesses a well-controlled manufacturing process and rigorous quality control standards (Gao et al., 2020).Preparation of XFC extract: Accurately weigh 4.0 g of XFC powder and transfer it to a stoppered conical flask (100 mL) containing 40 mL of methanol. Perform ultrasonic extraction at room temperature for 40 minutes, followed by filtration through filter paper. Concentrate the filtrate to dryness under reduced pressure, then reconstitute in 2 mL of methanol. Centrifuge the resulting solution at 12,000 rpm for 10 minutes at 4 °C, collect the supernatant, and filter it through a 0.22 μm nylon membrane filter. All prepared solutions should be stored at 4 °C and protected from light.LC-MS Analysis: LC separation was performed using a Thermo Vanquish ultra-high performance liquid chromatography system (Thermo Fisher Scientific, USA) equipped with an ACQUITY UPLC® HSS T3 column (2.1 × 150 mm, 1.8 µm) (Waters, Milford, MA, USA). The mobile phase consisted of acetonitrile and water containing 0.1% formic acid, and gradient elution was carried out at a flow rate of 0.25 mL/min. The column temperature was maintained at 40 °C, and the injection volume was 2 μL. Mass spectrometry analysis was conducted using an electrospray ionization (ESI) source in both positive and negative ion modes. ESI source parameters were as follows: sheath gas flow rate, 30 arb. units; auxiliary gas flow rate, 10 arb. units; positive ion spray voltage, 3.50 kV; negative ion spray voltage, -2.50 kV; capillary temperature, 325 °C. Full-scan MS was performed in the first stage at a resolution of 60,000 over a mass range of m/z 100-1000. For tandem mass analysis, HCD fragmentation was employed with a collision energy of 30%, secondary resolution of 15,000, and the top four precursor ions selected for fragmentation prior to signal acquisition. Dynamic exclusion was applied to eliminate redundant MS/MS spectra.The detailed chromatographic analysis conditions are provided in the supplementary materials.The high and low doses of XFC used in this study (648 mg/kg/day and 162 mg/kg/day, respectively) were determined according to the body surface area conversion principle commonly applied in pharmacological studies. Based on this method, the conversion factor between rats and humans is 6.3, yielding a rat-equivalent daily dose of 324 mg/kg/day. The high and low doses were set at twice and half this value, respectively.After 1 week of adaptive feeding, 36 Sprague-Dawley (SD) rats were included in the study and randomly assigned to the following groups: Control, AA model, XFC treatment (high-and low-dose, H-XFC and L-XFC), and Leflunomide (LEF) positive control group. Each group consisted of six rats. The AA model was established as described previously (Wan et al., 2013), after which treatment commenced. XFC was ground into a fine powder and formulated into 0.068 g/mL (H-XFC) and 0.017 g/mL (L-XFC) suspensions for intragastric administration in the XFC group. LEF was similarly prepared into a 0.095 mg/mL suspension. The AA group received intragastric administration of 0.9% normal saline. All groups received a daily intragastric dose of 1 mL/100 g body weight at 2 pm for 4 weeks. This study was approved by the Animal Ethics Committee of Anhui University of Traditional Chinese Medicine (Approval No. AHUCM-rats-2021081).The joint swelling of the rat (paw volume) and arthritis scores were recorded every 5 days.Twenty-four hours after the final administration, rats were anesthetized via intraperitoneal injection of pentobarbital, and the lung function was assessed using an animal lung function apparatus. The trachea was rapidly isolated, and bronchoalveolar lavage was performed with 10 mL/kg of normal saline, repeated three times. The lavage fluid was collected and centrifuged at 1500 r/min for 10 minutes at 4°C, and the supernatant was transferred into Eppendorf (EP) tubes and stored at -80℃. Finally, rats were euthanized, and lung and joint synovial tissues were collected.The left ankle joint synovial tissue and lung tissue were embedded in paraffin and sectioned at 5 μm thickness. Sections of both tissues were stained with H&E, while lung tissue sections were also stained with Masson's staining. Pathological changes of joint synovial and lung tissues were observed under an optical microscope. 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The of and were using analyses were performed using the for to analysis of the of and of were was using the and of was using For that these was followed by of was followed by the was were expressed as was were using the after Freund's complete adjuvant rats swelling of the the the arthritis (AA) model was A was then via injection of mL of at the of the volume and arthritis scores were recorded with the control the AA group showed volume and arthritis which on after 2 AA rats received different doses of XFC or LEF for therapeutic intervention. with the AA both LEF and H-XFC reduced swelling and arthritis scores on also showed significant only from significant differences were observed between the and groups analysis of joint synovial tissue in AA rats revealed synovial proliferation and cell with XFC or LEF reduced synovial and These findings that H-XFC exerts therapeutic efficacy H-XFC was selected as the dosage for experiments the effects of XFC on lung injury by pulmonary and in AA rats. model rats received XFC or high or LEF for with the AA both H-XFC and LEF treatment reduced cell and in lung were also observed in the characterized by a and significant differences were observed between the H-XFC and LEF treatment not pathological changes to the AA H-XFC and LEF also the of fibrosis and and reduced of and in bronchoalveolar lavage fluid and In addition, pulmonary function and improved in both groups and significant differences were between H-XFC and while produced investigate the potential targets of XFC in lung we performed proteomic analysis of lung tissues from AA rats. A of proteins were of which the for a of and we identified in rats with the AA group. proteins were and 36 were The top were and while the top were and changes were in a and a the and with the identified and analyses were to the between the was to identify analysis was conducted in three and In significant changes were observed in cellular to cellular to oxidative stress, cellular to chemical stress, cell and of In changes were in of the of the of the and In the most affected included on or groups of with or as regulator cell and transforming growth factor receptor These results that XFC lung injury in AA rats by cell processes and oxidative pathway analysis revealed the with in the XFC group. The most included the signaling the signaling chemical and Among the signaling which is to oxidative stress, was with and HMGCS2 was the most analysis of was conducted using to identify potential XFC targets in the treatment of lung injury in AA rats. The resulting which were key proteins for Notably, HMGCS2 and both with oxidative stress were and in the signaling pathway These findings that XFC lung injury in AA rats by modulating the signaling pathway to oxidative determine XFC alleviates lung injury by oxidative stress through the signaling we changes in proteins and oxidative stress following XFC intervention. with the AA the of PPARγ in the XFC group was significant was observed in These results that XFC the signaling pathway through PPARγ the of and were in the XFC group that XFC the of in lung tissue while the of the confirm that XFC and oxidative stress to pulmonary fibrosis by the PPARγ signaling an in vitro cell model was established using PPARγ were while and were the of the treatment of with a PPARγ 10 for PPARγ while the of and In addition, with PPARγ at 5 and serum for hours demonstrated that serum the and oxidative effects of this treatment PPARγ and reduced the of and In the serum has to is a autoimmune characterized by with joint RA is frequently by extraarticular with pulmonary common and studies that of RA patients form of lung with ILD representing of the most and complications, in to of et al., et al., as which often be through and pulmonary function In the disease to interstitial a major to and et al., and are therefore are often delayed or pharmacological of anti-fibrotic and novel et al., However, of and is with significant including and as well as the of et al., addition, anti-fibrotic and are and a considerable economic et al., These an clinical need for and treatment TCM has attention as a potential therapeutic approach for et al., TCM of that through diverse offering potential in as to Professor of Anhui University of lung injury is in TCM to which leads to the of and in the as and Based on this the XFC was XFC is a TCM that has been over the clinical and provide evidence its efficacy in lung A trial demonstrated that XFC not only joint symptoms but also improved pulmonary and in patients with RA animal studies that XFC lung function in arthritis (AA) rats, through of regulatory and the signaling pathway (Wan et al., Liu, Wang, & 2014). However, its effects on interstitial fibrosis and the underlying mechanisms remain this study, we employed and proteomic to investigate the therapeutic mechanisms of XFC in an AA rat model of RA-associated lung analysis revealed that the of XFC and et al., & Huang et al., et al., et al., et al., et al., et al., et al., et al., & et al., Li et al., et al., et al., of which are for and & Liu, and et al., are been to and effects in the AA These findings that the pharmacological of XFC is mediated by on a the effects of XFC on RA-associated lung an AA rat model Wang, & was established using and XFC treatments were LEF was used as a positive control due to its efficacy in oxidative stress, inflammation, and fibrosis et al., results showed that AA rats significant lung injury after 30 characterized by lung cell and of and and XFC treatment reduced arthritis and with the high dose the most Furthermore, XFC improved lung damage and in bronchoalveolar lavage and These results that XFC lung injury in AA rats, by and analysis of lung tissues identified between the and model Among HMGCS2 was and analyses that the most affected and included cellular to cellular to oxidative stress, and the signaling analysis identified HMGCS2 and as role in the therapeutic of stress is a key of and lung injury et al., et al., 2022). pulmonary and and to tissue et al., 2017). and are major of & & 2020). is in pulmonary fibroblasts and its fibrosis in et al., 2022). is a critical enzyme that oxidative et al., & proliferator-activated are that diverse processes & and is expressed in the and In PPARγ is widely expressed in lung and & et al., has not been in lung PPARγ be by as or which and PPARγ et al., and the of and in & In pulmonary fibrosis PPARγ been shown to weight and & 2024). also et al., and reduce in et al., 2014). PPARγ by through et al., the of and to form a key in its has been shown to HMGCS2 in and et al., & cell and by of et al., 2017). signaling et al., findings that XFC PPARγ resulting in the of a in oxidative stress, and the of and This is with studies on PPARγ as which shown efficacy in of lung fibrosis et al., et al., et al., However, clinical of PPARγ is by including weight fluid and & XFC, with its a both XFC and PPARγ as a common analysis that XFC a of bioactive and with and et al., et al., et al., et al., and XFC to the pathway in a and while additional anti-fibrotic and This is of TCM and provide a and therapeutic approach for complex conditions as RA-associated lung the TCM by Professor RA-associated lung injury from to with our molecular In the and its of oxidative stress and and of be as and The of and and reduced by XFC PPARγ as a between TCM and PPARγ and XFC the TCM of and its pathological this study that XFC PPARγ and oxidative stress as well as in AA rat Furthermore, using a pulmonary fibroblast model, we demonstrated that XFC fibrosis through of and oxidative the AA rat model is widely used in experimental research, it not the complex and pathological of lung which the of our findings into clinical while our by proteomic the involvement of the PPARγ/HMGCS2 pathway in oxidative stress and experiments HMGCS2 or be to a between this pathway and the observed as well as to its role in the of lung injury in AA rats. the of this study our ability to changes Finally, the of the bioactive components XFC and the of its therapeutic effects remain to be the effects of XFC on pulmonary fibrosis and is a key of our and We are an study in animal and to the of XFC on pulmonary fibrosis with RA in Despite these our findings provide a for and potential clinical our study that XFC lung injury in AA rats and and oxidative stress proteomic analysis, with in vitro and in vivo the molecular of XFC was We