Abstract

Purpose

   To investigate the effectiveness of modified Bu-Shen-Yi-Qi decoction
   (MBSYQ) in the treatment of osteoporosis associated with chronic
   obstructive pulmonary disease (COPD) and its underlying mechanisms of
   action.

Methods

   Disease targets, active ingredients and targets were predicted by TTD,
   CTD, DisGeNET, HERB (BenCaoZuJian as its Chinese name), and
   multiple-TCM databases; In addition, the screened targets were
   performed via the online platforms DAVID 6.8 and Metascape for GO and
   KEGG pathway enrichment analysis; The relationship between the MBSYQ
   and core targets were verified by molecular docking technique. Then we
   established a COPD-associated osteoporosis rat model by passive 24-week
   cigarette exposure. We assessed the efficacy of MBSYQ by lung
   histopathology assessment and distal femur/the first lumbar vertebra
   (L1) microstructural assay. In addition, we performed tibial RNA
   sequencing, which was validated by RT-PCR and Western blot.

Results

   Screening revealed that the 350 active compounds of MBSYQ anchored 228
   therapeutic targets for COPD-related osteoporosis; KEGG pathway
   enrichment analysis showed that the key targets mainly regulated MAPK
   and PI3K/AKT signaling pathways. In vivo studies showed that MBSYQ
   treatment alleviated pathological alterations in lung tissue, and
   reversed the bone loss and microstructure damage in the femur/L1 of
   model rats. The RNA seq indicated that MBSYQ could upregulate genes
   associated with anti-oxidative stress and aerobic respiration. The GSEA
   analysis displayed that MAPK and PI3K/AKT pathways were inhibited by CS
   exposure and activated by MBSYQ.

Conclusion

   MBSYQ is effective in the prevention and treatment of COPD-related
   osteoporosis, partially achieved by improving oxygen metabolism and
   activating MAPK and PI3K/AKT pathways.

   Keywords: COPD, osteoporosis, modified Bu-Shen-Yi-Qi formulae, network
   pharmacology, transcriptomics

Introduction

   Data show that the third leading cause of death worldwide is caused by
   chronic obstructive pulmonary disease (COPD).[36]1 It’s a common,
   preventable and treatable chronic respiratory disease characterized by
   incomplete reversibility and progressive airflow limitation. COPD is
   often associated with comorbidities and systemic consequences,
   including lung cancer, muscle weakness, osteoporosis, and further
   decreases the quality of life and increases mortality.[37]2–4 Patients
   with COPD are significantly more likely to develop osteoporosis than
   healthy individuals, and its severity is strongly associated with a
   decrease in bone mineral density (BMD).[38]5–10 In addition, the most
   common cause of secondary osteoporosis in men may be due to COPD.[39]11

   Chronic airway inflammation, characterized by inflammatory cell
   infiltration and increased secretion of inflammatory mediators, is
   thought to be the main cause of the ongoing progression of COPD.[40]12
   Osteoporosis is generally characterized by low bone mass,
   microarchitectural degeneration, and increased fracture risk.[41]13
   Although the prevalence of osteoporosis is significantly increased in
   patients with COPD compared to normal people, the causal and molecular
   links between them remain unclear.[42]14 It is difficult to clarify the
   pathological mechanism because of the various factors that affect bone
   metabolisms, such as smoking, weight, COPD acute exacerbation,
   sarcopenia, steroid use, etc.[43]15 To make matters worse, COPD-related
   osteoporosis is highly neglected by respiratory and physicians, and no
   formal guidelines for managing COPD-associated
   osteoporosis.[44]2,[45]16,[46]17 Due to the paucity of specific
   evidence in COPD patients, treatment is generally guided by the
   practice guidelines for primary osteoporosis. Current treatments
   include interventions for patients’ lifestyles (moderate exercise,
   healthy diet, etc.) and pharmacological treatments (calcium and vitamin
   D supplementation, etc.), selective estrogen receptor modulators, RANKL
   inhibitors, calcitonin, bisphosphonates, and PTH receptor agonists,
   etc.[47]18 However, it’s limited in its efficiency in restoring bone
   loss or increasing bone mass and has unavoidable side effects.[48]19
   Mesenchymal stem cell transplantation is thought to be a novel
   treatment for osteoporosis. However, it is still in preclinical studies
   and clinical trials, and its safety and efficacy need to be further
   evaluated.[49]20,[50]21

   Traditional Chinese Medicine (TCM) is an integrated system of medicine
   that has played an important role in the maintenance of people’s health
   worldwide for thousands of years.[51]22,[52]23 The efficacy of TCM has
   been proven to be a viable alternative therapy for the treatment of
   diseases.[53]24 Bu-Shen-Yi-Qi decoction, composed of three herbs
   including Epimedium brevicornum Maxim. (Epimedii Herba), Astragalus
   mongholicus Bunge (Radix Astragali), and Rehmannia glutinosa (Gaertn.)
   DC. (Radix Rehmanniae), have been proven effective in clinical and
   experimental studies for the treatment of chronic respiratory diseases
   such as asthma and COPD.[54]25–28 Our previous randomized,
   double-blinded, multicenter clinical trial demonstrated that it
   improved lung function, reduced the frequency of acute exacerbations,
   and attenuated the inflammation response in COPD patients.[55]27 In
   addition, a modified version of the Bu-Shen-Yi-Qi formulae (MBSYQ,
   Scutellaria baicalensis Georgi (Radix Scutellariae) and Paeonia
   lactiflora Pall. (Radix Paeoniae Rubra) were added, detailed in
   [56]Table S1) is more effective in treating acute exacerbations of
   COPD, such as shortness of breath and relief of cough.[57]29 However,
   it remains unclear whether MBSYQ is effective in the prevention and
   treatment of COPD-related osteoporosis.

   We first explored the potential molecule link between COPD and
   osteoporosis from the perspective of network pharmacology, the possible
   mechanisms of MBSYQ for COPD-related osteoporosis treatment, and then
   established a 24-week CS-induced animal model to validate the
   pathological mechanisms, influencing factors and underlying mechanisms
   of MBSYQ for COPD-related osteoporosis treatment, which can provide
   further clinical applications and basic research. The study design is
   displayed in [58]Figure 1

Figure 1.

   [59]Figure 1
   [60]Open in a new tab

   Schematic diagram of the study to explore the pharmacological
   mechanisms of MBSYQ in COPD-related osteoporosis.

Materials and Methods

COPD & Osteoporosis-Related Protein Screening

   Therapeutic Target Database (TTD,
   [61]http://bidd.nus.edu.sg/group/cjttd/)[62]30 and Comparative
   Toxicogenomics Database (CTD, [63]http://ctdbase.org/)[64]31 can be
   used to screen target proteins for COPD and osteoporosis; Potential
   targets for COPD and osteoporosis were collected by DisGeNET database
   ([65]https://www.disgenet.org/).[66]32 We searched all three databases
   using the keywords “Chronic obstructive pulmonary disease” or
   “Osteoporosis” and set the species to “Homo sapiens”. Finally, we
   removed duplicates and integrated all information to further analyze
   proteins common to COPD and osteoporosis.

Bioactive Ingredients Collection and Target Prediction

   Potentially active compound screening and candidate target prediction
   for MBSYQ were screened from the HERB database (BenCaoZuJian as its
   Chinese name, obtained from [67]http://herb.ac.cn/) which links 12,933
   targets and 28,212 diseases to 7263 herbal medicines and 49,258
   ingredients. They provided six pairwise relationships between them by
   integrating multiple-TCM databases (SymMap, TCMID 2.0, TCMSP 2.3, and
   TCM-ID) and manually 1966 reference comparisons.[68]33 We further
   searched SuperTarget
   ([69]https://bioinformatics.charite.de/supertarget/),[70]34
   SwissTargetPrediction ([71]www.swisstargetprediction.ch),[72]35
   Similarity ensemble approach (SEA, [73]https://sea.bkslab.org/),[74]36
   STICH ([75]http://stitch.embl.de/)[76]37 and PharmMapper
   ([77]http://www.lilab-ecust.cn/pharmmapper/)[78]38 databases for
   component-target prediction of icaritin and astragaloside IV. Networks
   were built and visualized with Cytoscape 3.8.0.[79]39 Network
   parameters were calculated by the NetworkAnalyzer plugin.

Gene Ontology (GO) and Pathway Enrichment Analysis

   To further reveal the mechanism of MBSYQ for COPD-related osteoporosis,
   through the online platforms DAVID 6.8
   ([80]https://david.ncifcrf.gov/)[81]40 and Metascape
   ([82]https://metascape.org/),[83]41 the targets of MBSYQ for
   COPD-related osteoporosis therapy were additionally analyzed by GO and
   KEGG pathway enrichment.

Molecule Docking

   Three-dimensional chemical structures of the core active ingredients
   were obtained from the PubChem database (NCBI, USA), and energy
   minimized by molecular mechanics-2 (MM2) force fields in Chem 3D Pro;
   The RCSB Protein Data Bank ([84]www.rcsb.org) was used to retrieve the
   crystal structures of the target proteins; The AutoDock tool (1.5.6)
   was performed for the addition of hydrogen atoms and the removal of
   water and heterogeneous molecule; AutoDock vina was employed to predict
   potential molecular binding patterns between components and candidate
   targets; the docked structures were analyzed via PyMol 2.3.0
   ([85]http://www.pymol.org/). The position of the original ligand
   defines the center of the active site for protein-ligand docking, set
   to a grid size of 40 × 40×40 Ǻ in the x, y, and z directions, with the
   spacing between the two grid points set to 0.375 Ǻ. The most likely
   binding mode is the docking conformation corresponding to the lowest
   binding energy.

Chemicals and Reagents

   Primary antibodies against P-JNK (4668T), P-ERK (4370), P-P38 (4511T),
   and β-actin (4970) were from Cell Signalling Technology (Danvers, USA).
   Anti-P-Akt (66444-1-Ig) was obtained from Proteintech. Commercial
   tobacco Daqianmen was purchased from Shanghai Jieqiang Tobacco, Sugar
   and Wine (Group) Co., Ltd.

MBSYQ Preparation

   MBSYQ granules were purchased from Jiangyin Tianjiang Pharmaceutical
   Co., Ltd. Briefly, the herbs were decocted twice with water, then
   filtered and concentrated to a relative density of 1.11–1.13
   (Astragalus mongholicus Bunge), 1.09–1.11 (Epimedium brevicornu
   Maxim.), 1.09–1.12 (Rehmannia glutinosa (Gaertn.) DC.), 1.15–1.17
   (Scutellaria baicalensis Georgi) and 1.05–1.07 (Paeonia lactiflora
   Pall.) at 65±5°C, respectively. The herbal extracts were obtained by
   spray drying with 18–40 mesh screening and then quality controlled by
   ultraviolet-visible spectrophotometry (UU-Vis) and high-performance
   liquid chromatography (HPLC). The pellets of each herb were mixed in
   proportion and diluted with potable water before administration.

Animal Model and Treatment

   Selected 40 male SD rats (6 weeks old, 160–200 g) (Sippr-BK Laboratory
   Animals Co. Ltd., Shanghai, China) and housed in a specific
   pathogen-free (SPF) condition under a 12: 12 h light-dark cycle and
   given free access to food and water. Animal experiments were approved
   by the Animal Experimentation Ethics Committee of Fudan University
   (Approval No. 202011005S). Rats were acclimated to the conditions for
   one week before the experiment and randomly divided into five groups
   (n = 8 per group): Normal control group, CS group, CS + MBSYQ
   low/medium/high dose group (6.25 g/kg, 12.5 g/kg, 25 g/kg). The medium
   doses given to rats were calculated based on the animal dose conversion
   table and body surface area.[86]42 Construction of rat model of chronic
   obstructive pulmonary disease with slight modifications from
   references.[87]43–45 Briefly, rats were placed in a homemade acrylic