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Back to Journal »Journal of Inflammation Research» Volume 14
Sahastraputi-Abhrak-Bhasma, a traditional Indian medicine based on biotite calcium, prevents allergic airway inflammation in a mouse model of asthma by modifying the cytokine response
Authors: Balkrishna A, Solleti SK, Singh H, Singh R, Sharma N, Varshney A
Published on September 17, 2021, the 2021 volume: 14 pages 4743-4760
DOI https://doi.org/10.2147/JIR.S313955
Single anonymous peer review
Reviewing editor: Professor Quan Ning
Acharya Balkrishna, 1--3 Siva Kumar Solleti, 1, * Hoshiyar Singh, 1, * Rani Singh, 1 Niti Sharma, 1 Anurag Varshney 1, 2, 4 1 Haridwa Patanjali Institute of Medicine, Uttarakhand, India Discovery and Development Department; 2 Department of Joint and Applied Sciences, Patanjali University, Patanjali Yog Peeth, Haridwar, Uttarakhand, India; 3 Patanjali UK Trust, Glasgow, UK; 4 Jawaharlal, New Delhi, India Special Center for Systems Medicine, Nehru University* These authors contributed equally to this work 91 1334-244107 x7458 Fax 91 1334 244805 Email [email protected] Purpose: Asthma is a heterogeneous airway inflammation Sexual diseases, treatment options are limited. Traditional medicine is widely used to treat various diseases including asthma. Sahastraputi-Abhrak-Bhasma (SPAB) is an Indian medicine based on biotite calcium. Methods: We tested the anti-inflammatory and anti-asthmatic properties of SPAB using ovalbumin-induced in vivo models of allergic asthma in mice and in vitro cell analysis. Histological analysis, qPCR and ELISA were performed to assess pathology. SEM, EDX and XRD analysis were performed to characterize SPAB particles. Results: SEM, EDX and XRD analysis confirmed the presence of SPAB particles with a diameter of 100 nm to 1 μm. The sample contained anniite-1M, aluminum silicate, kyanite, alumina, magnesium silicate and maghemite. Ova-challenge causes severe inflammation, airway remodeling and increased lung oxidative load. Importantly, the preventive treatment of SPAB significantly reduced allergen-induced leukocyte infiltration, especially eosinophils, lymphocytes, macrophages and neutrophils in BALF. SPAB treatment significantly eliminated egg-induced mucus hypersecretion, peribronchial collagen deposition, inflammatory cell infiltration, and bronchial epithelial thickening. qPCR and ELISA analysis confirmed the allergen-induced increase in IL-5, IL-13, IL-33, IFN-γ and IL-1β cytokine mRNA and IL-6, IL-1β and TNF-α protein levels in the whole lung After SPAB treatment, BALF in BALF was significantly weakened. SPAB restored the allergen-induced reduction of antioxidant markers in the lung. In vitro, SPAB attenuated the secretion of IL-6 and TNF-α in human bronchial epithelial cells, and moderately inhibited the NF-kB/AP-1 pathway in HEK cells. Conclusion: In general, our results verified the potential of the classic Indian drug Sahastraputi-Abhrak-Bhasma to prevent and improve asthma-related airway inflammation through experiments. Keywords: Sahastraputi-Abhrak-Bhasma, asthma, physical and chemical analysis, lung inflammation, cytokines, mica drugs
Asthma is a common airway heterogeneous disease, affecting millions of people worldwide, and its incidence is rising. 1,2 It is characterized by inflammation of the lung airways, wheezing, coughing, and shortness of breath. 3 Allergic asthma is more common in developing countries and is associated with Th2 cell response. 2 It includes the secretion of Th2 related cytokines, such as interleukin (IL)-4, IL-5 and IL-13.3, which in turn leads to Eosinophilic airway inflammation, airway hyperresponsiveness (AHR), mucus hypersecretion, and airway wall remodeling. 4 AHR can cause airflow obstruction. In clinical practice, long-term use of steroidal anti-inflammatory drugs to treat asthma can lead to potential complications, such as pneumonia and hyperglycemia. 5
In the pathology of asthma, the participation of eosinophils, mast cells, dendritic cells, lymphocytes, macrophages and neutrophils and other cells is obvious; immune cells and structural cells (such as epithelial cells and interstitial cells) The interaction between mesenchymal cells plays a crucial role in the progression and severity of the disease. 3,6 Although extensive progress has been made in the management of asthma and allergies, in nearly 5-10% of people, this condition continues to be severe and cannot be effectively controlled. 5 Currently, there is no established way to avoid asthma and allergies. Therefore, new treatment strategies for effective treatment must be determined.
The time importance of traditional medicine as a medicine for the treatment of asthma is irrefutable7. 60-70% of patients with moderate and severe asthma rely on traditional medicine as a supplement. 7Metal or herbal mineral therapy for the treatment of asthma has been put into practice. 8 The Rasa-shastra treatment of ancient Indian Ayurveda uses various incineration procedures to utilize the healing potential of various herbs, minerals, metals, and non-metals, transforming them into a non-toxic, biologically acceptable form called bhasma. 9,10 This has recently been defined by the WHO. 11 However, proper scientific verification of the therapeutic potential of bhasma is lacking.
Abhrak bhasma is a traditional Indian medicine based on biotite calcium, prepared through a process called puta (burning in a closed earth container) to take advantage of its unique properties. 12,13 Calx is defined as the ash or oxide material remaining after metals and minerals have been incinerated in an orderly manner. Shataputi Abhrak Bhasma and Sahastraputi-Abhrak-Bhasma (SPAB) are two unique preparations in which mica is titrated with different herbs, burned 100 times and 1000 times respectively, and then treated with tallow to produce mica/biotite calcium. Abhrak bhasma is classic for the treatment of anemia and tropical aphtha, chronic cough, bronchitis and asthma. 14 It can prevent various cancers, 13 CCl4 induces hepatitis, 15 improves kidney and liver function, 16 and exerts diuretic activity. 17
Shataputi abhrak bhasma and SPAB are considered the best biotite calcium (mica-based) bhasma14 and contain immunomodulatory properties. 18 SPAB has been shown to prevent heat-induced oligospermia and azoospermia, possibly by regulating p53 and Fas-mediated apoptosis pathways and improving germinal epithelial proliferation. 19 The purpose of this study is to scientifically verify the anti-inflammatory and anti-asthma properties of SPAB using a mouse model of allergic asthma induced by ovalbumin (Ova), and to analyze its chemical composition.
Sahastraputi-Abhrak-Bhasma (SPAB) is from Divya Pharmacy, Haridwar, India. Scanning electron microscope (SEM; LEO-438 VP) combined with electron dispersive X-ray analysis system (Carl Zeiss, Germany) is used to analyze the morphology and element characteristics of SPAB powder. SEM imaging was performed at an accelerating voltage of 10 kV. EDX analysis is done through mapping and point-based analysis. The X-ray diffraction (XRD) analysis of SPAB powder was performed using a Rigaku D-Max 2200 X-ray diffractometer (XRD) with Cu-Kα radiation at 40 kV/40 mA. Scanning with 0.02° steps in the angle range of 5° to 80°, the scan rate is 0.5° min-1.20 Use QualX (version 2.24) to qualitatively analyze the XRD spectrum of SPAB. twenty one
SPF-free female BALB/c mice weigh 20-22 grams at eight weeks of age and are kept in an animal terrarium under a light-dark cycle of 12:12 hours. The mice were purchased from Hylasco Biotechnology Pvt, an animal supplier licensed by Charles River Laboratory. Ltd., Hyderabad, India, using standard pellet diet (Purina Lab Diet, St. Louis, MO, USA) and sterile water ad libitum nourishment. Animal experiments and treatment procedures were approved by the Animal Ethics Committee of the Patanjali Institute (IAEC approval number-PRIAS/LAF/IAEC-078; date of approval: July 17, 2019) to comply with the guidelines and animals of the Control Purpose Committee Experimental Supervision (CPCSEA), Government of India.
For in vivo experiments, the mouse equivalent dose of SPAB was calculated based on the body surface area of the mouse. The recommended human therapeutic dose of SPAB is 500 mg/day, and the mouse equivalent dose (mg/kg) is calculated by multiplying the human equivalent dose (mg/kg) by 12.3.22. The calculated mouse therapeutic equivalent dose is 102. mg/kg is regarded as a medium dose (1x), and its 1/3x and 3x doses are regarded as low and high doses for pharmacological studies (34 mg/kg, 102 mg/kg and 306 mg/kg) .
Mice were assigned to six different treatment groups: (1) normal control (NC); (2) disease control (DC); (3) dexamethasone (DM)-2 mg/kg; (4) SPAB- 34 mg/kg; (5) SPAB-102mg/kg; (6) SPAB-306mg/kg. To establish an asthma model, (the mice in groups 2 to 6 were treated with ovalbumin (Ova) (25 μg) and aluminum hydroxide (2 mg) in 0.2 mL saline (PBS) on days 0, 7 and 14 Intraperitoneal (ip) sensitization as described above 23. On days 21, 23, 25, and 27, mice were challenged with 100 μg Ova-in PBS (25 μL) under mild anesthesia by intranasal infusion. Mice in the NC group received only PBS during sensitization and challenge. NC and DC received 0.5% carboxymethyl cellulose (CMC). From day 7 onwards, three different doses of 0.5% CMC were taken orally in a preventive way every day. SPAB. The DM group was intraperitoneally injected with 2 mg/kg DM, 2 hours before Ova challenge. Ovalbumin (Ova) and dexamethasone (DM) were from Sigma Aldrich (St. Louis, Missouri, USA). Figure 1 Physicochemistry of SPAB Analysis. (A) SPAB's scanning electron microscope (SEM) analysis confirmed the presence of particles of different shapes and (B) SPAB's electron dispersive X-ray (EDX) analysis showed the presence of oxygen (O), silicon (Si), and iron (Fe) , Aluminum (Al), potassium (K), magnesium (Mg), chlorine (Cl), nickel (Ni), chromium (Cr), sodium (Na), calcium (Ca) elements, with different content (illustration). (C) X-ray diffraction (XRD) analysis confirmed the presence of Annite-1M, aluminum magnesium, aluminum silicate, kyanite, alumina, magnesium silicate, and maghemite in the SPAB sample.
Figure 1 Physical and chemical analysis of SPAB. (A) SPAB's scanning electron microscope (SEM) analysis confirmed the presence of particles of different shapes and sizes. (B) SPAB's electronic dispersive X-ray (EDX) analysis shows the presence of oxygen (O), silicon (Si), iron (Fe), aluminum (Al), potassium (K), magnesium (Mg), chlorine (Cl), Nickel (Ni), chromium (Cr), sodium (Na), and calcium (Ca) elements have different contents (illustration). (C) X-ray diffraction (XRD) analysis confirmed the presence of Annite-1M, aluminum magnesium, aluminum silicate, kyanite, alumina, magnesium silicate, and maghemite in the SPAB sample.
Collect the BAL fluid and excise the lung lobes as previously described 23-25, and then manually calculate the total cell count and the differential cell count. In short, the mice were sacrificed, the left lung was ligated, and the right lung was lavaged with 3 volumes (1 mL/25 g) of ice-cold PBS by inserting a catheter in the trachea. 24 The resulting bronchoalveolar lavage fluid (BAL) was fluidly centrifuged (3000 rpm, 4°C for 3 minutes), and the supernatant was retained for further analysis. 1% acetic acid was used to lyse the red blood cells in the cell pellet and resuspend the BAL cells in PBS. The total cell count was measured, and a cell smear was prepared and stained with Wright staining. 24 Subsequently, 300 cells were classified using standard morphological criteria for a white blood cell count.
The histological examination was performed as described above. 23,25 In short, the left lung lobe was sectioned and stained with hematoxylin and eosin (H&E) for gross pathological analysis. Periodic-Acid-Schiff (PAS) staining was used to identify mucus secretion, and Mason-Trichrome (MT) staining was used to assess peribronchial collagen deposition. The severity of histopathological changes is determined using the following 5-point scoring method26,27: 0=no abnormality detected (NAD), 1=slight, 2=mild, 3=moderate, 4=moderately severe, 5= Severe; distribution is recorded as focal, multifocal, and diffuse.
Isolate total RNA from lung tissue using TRIzol reagent (Invitrogen, USA). As mentioned above, use Verso cDNA Synthesis Kit (Thermo Fisher Scientific, USA) for cDNA synthesis. 23,25 Make RNA free of DNA and repurify it for use RNeasy mini kit (Qiagen, USA) and reverse transcription of RNA using Verso cDNA synthesis kit (Thermo Fisher Scientific, USA). Quantitative real-time PCR was performed using Biometra T Professional thermal cycler (Analytik Jena, Germany) and SYBR green master mix (Applied Biosystems, USA). Use 2-ΔΔCt to calculate the gene expression level relative to Ppia (cyclophilin A) using primer sequences derived from the MGH primer library as described above 24,28.
According to Rotruck et al. 197329, DTNB reagent is used to measure glutathione peroxidase (GPX) activity in a 96-well format at 420 nm. Goth 199130 is suitable for measuring catalase (CAT) activity at 405 nm. Use NBT31 to measure superoxide dismutase (SOD) activity at 560 nm. Glutathione (GSH) and glutathione disulfide (GSSG) levels were measured by the protocol of Hissin & Hilf, 197632 at Ex 350/Em 420 nm fluorescence. According to Heath & Packer, 196833, measure the level of malondialdehyde (MDA) and measure the absorbance at 532 nm. To measure myeloperoxidase (MPO)/eosinophil peroxidase (EPO) activity, use 3,3ʹ,5,5ʹ-tetramethylbenzidine substrate 34,35 and at 450 nm as described Measure absorbance at each place. 36 The absorbance or fluorescence intensity was measured with Envision microplate reader (Perkin Elmer, USA) and expressed as U/mg or µM/mg lung tissue weight or U/mL BAL solution.
Perform an enzyme-linked immunosorbent assay (ELISA) of BALF to measure the levels of mouse IL-6, IL-1β, and TNF-α (BioLegend Inc., San Diego, USA) and the secretion of cytokines by BEAS2B into the culture The supernatant was measured using human IL-6 and TNF-α ELISA (BD Bioscience, USA) according to the manufacturer's instructions, and the plate was read at 450 nm using an Envision microplate reader (Perkin Elmer, USA).
The cell lines used in this study include BEAS2B cells (kindly provided by Dr. Rajarshi Pal, India, and approved for use by the Institutional Biosafety Committee of the Patanjali Institute) and HEK-Blue TNF-α cells (InvivoGen, USA). BEAS2B is stored in RPMI-1640 (Invitrogen, USA) containing 10% heat-inactivated FBS, L-glutamine (4 mM) and antibiotics. HEK-Blue TNF-α cells (stable expression of secreted embryonic alkaline phosphatase (SEAP) reporter gene fused with NF-κB/AP-1 elements) contained 10% heat-inactivated FBS, 100 μg/mL Normocin, L -Glutamine is cultured in DMEM (2 mM), 100 μg/mL streptomycin and 100 U/mL penicillin, and the cells are kept at 37°C in a humidified incubator with 5% CO2.
BEAS2B cells were seeded in 96-well plates at 1×105 cells/mL, and after reaching 70% confluence, they were treated with different doses of SPAB (1 µg/mL-100 µg/mL) for 24 hours. The cells were washed and incubated with 10 µL Alamar blue (0.15 mg/mL) (Hi Media, India) in serum-free medium for 1 hour, and fluoresced at Ex. 530 nm/Em. Measure 590 nm and express it as a percentage of cell viability. BEAS2B cells were pretreated with different concentrations of SPAB (1–30 µg/mL) for 24 hours, and then treated with SPAB 500 ng/mL lipopolysaccharide (LPS) for another 24 hours. The cell culture supernatant was used to measure cytokine secretion.
HEK-Blue TNF-α cells were seeded in a 96-well plate at a density of 3×105 cells/mL, and treated with different concentrations of SPAB (1 μg/mL to 10 μg/mL) for 6 hours, and then 500 pg/mL was added Human TNF-α. After 24 hours of incubation, the alkaline phosphatase activity was measured at 630 nm using QUANTI-Blue substrate according to the manufacturer's instructions (InvivoGen, San Diego, California, USA).
Statistical analysis was performed using GraphPad Prism version 7.0 software (GraphPad Software, San Diego, CA, USA). One-way analysis of variance (ANOVA) and Dunnett's multiple comparison test were used to calculate statistical differences. All data are expressed as mean ± standard error of mean (SEM). The difference in the mean is considered significant when p <0.05.
Scanning electron microscope (SEM) analysis of SPAB powder revealed the heterogeneity of SPAB in shape and size (Figure 1A) ranging from 100 nm to ~1 µm in diameter (Figure 1A). SPAB’s electronic dispersive X-ray (EDX) quantitative analysis showed the presence of oxygen (O) (55.5 ± 2.9%); silicon (Si) (6.8 ± 0.4%); iron (Fe) (18.1 ± 1.1%); aluminum (Al) (1.3±0.09%); Potassium (K) (2.2 ± 0.1%); Magnesium (Mg) (6.7 ± 0.4%); Chlorine (Cl) (0.43 ± 0.02%); Nickel (Ni) (2.7 ± 0.1%) ; Chromium (Cr) (2.9 ± 0.1%); Sodium (Na) (1.6 ± 0.1%); Calcium (Ca) (1.4 ± 0.09%) element on the surface of SPAB particles (Figure 1B). X-ray diffraction (XRD) analysis of SPAB found that there are various silica-rich compounds and aluminum, iron and magnesium compounds, such as Annite-1M (KFe32 AlSi3O10(OH,F)2), aluminum magnesium (Al-Mg) ), aluminum silicate (Al2SiO5), kyanite (Al2SiO5), alumina (Al2O3), magnesium silicate (MgO3Si) and maghemite (γ-Fe2O3) in the sample (Figure 1C). Figure 2 Oral SPAB treatment reduced Ova-induced leukocyte infiltration in the BAL fluid of asthmatic mice. (A) Schematic diagram of Ova-induced asthma study design. Mice were acclimated, randomized, sensitized and challenged with Ova-allergen. At the same time, as described in the method section, DM and various doses of SPAB were given and various end points were tested. 48 hours after the last Ova challenge, the total white blood cell count in the BAL fluid was quantified (B) the total eosinophils (C) the total eosinophils (D) Total lymphocytes, (E) total monocytes, (F) total neutrophils in BALF. Data are expressed as mean ± SEM (n = 4-6). **, ##p <0.001 and *p <0.05 are through one-way analysis of variance. ## means significant compared to NC, ** means significant compared to DC.
Figure 2 Oral SPAB treatment reduced Ova-induced leukocyte infiltration in the BAL fluid of asthmatic mice. (A) Schematic diagram of Ova-induced asthma study design. Mice were acclimated, randomized, sensitized and challenged with Ova-allergen. At the same time, as described in the method section, DM and various doses of SPAB were given and various end points were tested. 48 hours after the last Ova challenge, the total white blood cell count in the BAL fluid was quantified (B) the total eosinophils (C) the total eosinophils (D) Total lymphocytes, (E) total monocytes, (F) total neutrophils in BALF. Data are expressed as mean ± SEM (n = 4-6). **, ##p <0.001 and *p <0.05 are through one-way analysis of variance. ## means significant compared to NC, ** means significant compared to DC.
To determine whether SPAB can reduce allergen-induced airway inflammation, as described in the Materials and Methods section (Figure 2A), we first collected BALF and tested the total and differential leukocyte recruitment in the lungs. The results showed that the total number of inflammatory cells in DC lungs increased significantly after Ova sensitized and challenged (DC vs. NC, p<0.001) (Figure 2B), reflecting human clinical manifestations. In contrast, all three doses of SPAB significantly blocked the recruitment of inflammatory cells (DC vs. SPAB 34-306, p<0.001) (Figure 2B), similar to DM (DC vs. DM-2, p<0.001) (Figure 2B) 2B) Treatment group.
We next tested the differential cell count and found that the number of eosinophils, lymphocytes, macrophages, and neutrophils in DC increased significantly (DC vs. NC, p<0.001) (Figure 2C-F). It is worth noting that after daily oral SPAB administration (DC vs SPAB 34-306, p<0.001) in allergic mice (Figure 2C-F), the dose-dependent decrease in the number of cells mentioned above was noted, similar to DM (DC vs DM, p <0.001). These results indicate the beneficial effect of SPAB in regulating the influx of white blood cells induced by allergens into the lungs.
After confirming the inhibitory effect of SPAB on the recruitment of leukocytes in the lung, we next tested lung histopathology. PAS staining of lung tissue sections (Figure 3) was used for mucus secretion, and it was found that compared with NC, allergen-induced mucus accumulation in DC was significantly increased (Figure 3A, NC and DC), which increased the semi-quantitative airway mucus score ( Figure 3B). In contrast, allergic mice receiving three different doses of SPAB (Figure 3A, SPAB-34, -102, and -306) or DM (Figure 3A, DM-2) showed a significant decrease in mucus accumulation and airway mucus score (Figure 3B). Consistent with mucus accumulation, mRNA expression analysis confirmed that SPAB mediated a significant reduction in Muc5AC gene expression in allergic mice (DC vs SPAB 34-306, p<0.05) (Figure 3C), which was compared with DM-treated mice (DC vs DM-2, p<0.001). Figure 3 SPAB attenuates allergen-induced mucus hypersecretion through the airway epithelium. Assessment of mucus accumulation in PAS-stained lung tissue sections. (A) PAS stained lung tissue sections of NC, DC, DM, SPAB-34 mg/kg, SPAB-102 mg/kg and SPAB-306 mg/kg. (B) Quantitative histogram of mucus accumulation in airway epithelial cells. (C) Whole lung mRNA expression of Muc5AC gene. The black arrow indicates the bronchial epithelium, and the white arrow indicates the mucus-secreting airway epithelium. Data are expressed as mean ± SEM (n = 4-6). **, ##p <0.001 and *p <0.05 through one-way analysis of variance; ## represents significant compared with NC, * represents significant compared with DC (scale bar represents 20μm).
Figure 3 SPAB attenuates allergen-induced mucus hypersecretion through the airway epithelium. Assessment of mucus accumulation in PAS-stained lung tissue sections. (A) PAS stained lung tissue sections of NC, DC, DM, SPAB-34 mg/kg, SPAB-102 mg/kg and SPAB-306 mg/kg. (B) Quantitative histogram of mucus accumulation in airway epithelial cells. (C) Whole lung mRNA expression of Muc5AC gene. The black arrow indicates the bronchial epithelium, and the white arrow indicates the mucus-secreting airway epithelium. Data are expressed as mean ± SEM (n = 4-6). **, ##p <0.001 and *p <0.05 through one-way analysis of variance; ## represents significant compared with NC, * represents significant compared with DC (scale bar represents 20μm).
In addition, compared with unchallenged NC mice (Figure 4A, NC and DC), Masson's trichrome staining of lung tissue sections revealed a significant increase in peribronchial collagen deposition in allergic DC mice. This resulted in a significant increase in the collagen deposition score, measured semi-quantitatively (DC vs. NC, p <0.001) (Figure 4B). However, treatment with DM (Figure 4A, DM-2) or SPAB (Figure 4A, SPAB-34, -102 and -306) can inhibit collagen deposition and significantly reduce the collagen deposition score (DC vs. SPAB 34-306, p <0.001) (Figure 4B). Figure 4 Oral SPAB administration reduced peribronchial collagen deposition in allergic mice. Masson's trichrome staining of lung tissue sections was used to evaluate OVA-induced collagen deposition around the airways. (A) MT stained lung tissue sections of NC, DC, DM-2 mg/kg, SPAB-34 mg/kg, SPAB-102 mg/kg and SPAB-306 mg/kg. (B) Quantitative histogram of collagen accumulation around the bronchus. The black arrow points to the airway epithelium, and the white arrow indicates collagen deposition. Data are expressed as mean ± SEM (n = 4-6). **, ##p <0.001 one-way analysis of variance; ## represents significant compared with NC, ** represents significant compared with DC (scale bar represents 20μm).
Figure 4 Oral SPAB administration reduced peribronchial collagen deposition in allergic mice. Masson's trichrome staining of lung tissue sections was used to evaluate OVA-induced collagen deposition around the airways. (A) MT stained lung tissue sections of NC, DC, DM-2 mg/kg, SPAB-34 mg/kg, SPAB-102 mg/kg and SPAB-306 mg/kg. (B) Quantitative histogram of collagen accumulation around the bronchus. The black arrow points to the airway epithelium, and the white arrow indicates collagen deposition. Data are expressed as mean ± SEM (n = 4-6). **, ##p <0.001 one-way analysis of variance; ## represents significant compared with NC, ** represents significant compared with DC (scale bar represents 20μm).
We next used H&E staining to assess lung inflammation and determined peribronchial and perivascular leukocyte aggregates in DCs challenged by allergens compared with NC mice (Figure 5A, NC and DC), and leukocytes in lungs Recruitment also increased significantly (DC vs. NC, p<0.001) (Figure 5B). In contrast, treatment with DM (Figure 5A, DM-2) and SPAB (Figure 5A, SPAB-34, -102 and -306) significantly inhibited (p<0.05) inflammatory cell infiltration (Figure 5B) and reduced white blood cells Aggregation. In addition, allergen-induced bronchial epithelial thickening was significantly greater after treatment with SPAB (DC and SPAB 34-306, p<0.05) (Figure 5C) and DM (DC and DM-2, p<0.05) (Figure 5C). It is lowering. In general, after oral SPAB treatment, the increase in total pathology score caused by Ova allergen (p<0.001) (Figure 5D) was significantly cancelled (p<0.001). Figure 5 SPAB improves histopathological changes in airway inflammation. H&E staining was used to assess airway inflammation in lung tissue sections. (A) H&E stained lung tissue sections of NC, DC, DM-2 mg/kg, SPAB-34 mg/kg, SPAB-102 mg/kg and SPAB-306 mg/kg. (B) Quantitative analysis of leukocyte recruitment in the lung, (C) bronchial epithelial thickening and (D) total pathology score. The arrow points to accumulation of white blood cells or thickening of the bronchial wall. Data are expressed as mean ± SEM (n = 4-6). **, ##p <0.001 and *p <0.05 through one-way analysis of variance; ## means significant compared with NC, * means significant compared with DC (scale bars indicate 100 µm and 20 µm).
Figure 5 SPAB improves histopathological changes in airway inflammation. H&E staining was used to assess airway inflammation in lung tissue sections. (A) H&E stained lung tissue sections of NC, DC, DM-2 mg/kg, SPAB-34 mg/kg, SPAB-102 mg/kg and SPAB-306 mg/kg. (B) Quantitative analysis of leukocyte recruitment in the lung, (C) bronchial epithelial thickening and (D) total pathology score. The arrow points to accumulation of white blood cells or thickening of the bronchial wall. Data are expressed as mean ± SEM (n = 4-6). **, ##p <0.001 and *p <0.05 through one-way analysis of variance; ## means significant compared with NC, * means significant compared with DC (scale bars indicate 100 µm and 20 µm).
After confirming the reversal of the SPAB-mediated lung histopathological response, we tested the mRNA expression of asthma-related pro-inflammatory genes. The results showed that compared with the non-attacked NC allergen challenge in DC, the expression of interleukin (IL)-5 (p<0.001) (Figure 6A) and IL-13 were significant (DC vs. NC, p< 0.001) multiple up-regulation (p<0.001) (Figure 6B), IL-33 (p<0.001) (Figure 6C), interferon (IFN)-ɣ (Figure 6D) and IL-1β (p<0.001) (Figure 6C) 6E). Therapeutic administration of SPAB significantly attenuated Ova-induced IL-5 (p<0.05) (Figure 6A), IL-13 (p<0.05) (Figure 6B), IL-33 (p<0.001) (Figure 6C ), IFN-ɣ of medium to high dose or highest dose of IL-1β (Figure 6D) (p<0.05) (Figure 6E). In addition to IL-1β (NS) (Figure 6E), DM treatment of the above genes (Figure 6A-D) also resulted in a similar decrease in the expression of pro-inflammatory genes. Figure 6 Oral administration of SPAB reduced the levels of pro-inflammatory cytokines induced by OVA allergens. Steady-state mRNA expression levels of cytokines in whole lung tissue, (A) IL-5, (B) IL-13, (C) IL-33, (D) IFN-γ and (E) IL-1β. BAL Fluid tests (F) IL-1β, (G) TNF-α and (H) IL-6 and protein cytokine levels by ELISA. mRNA changes are expressed as relative fold changes, and cytokine levels are expressed as pg/mL BALF. Data are expressed as mean ± SEM (n = 4-6). **, ##p <0.001 and *p <0.05 are through one-way analysis of variance. ## means significant compared to NC, ** means significant compared to DC.
Figure 6 Oral administration of SPAB reduced the levels of pro-inflammatory cytokines induced by OVA allergens. Steady-state mRNA expression levels of cytokines in whole lung tissue, (A) IL-5, (B) IL-13, (C) IL-33, (D) IFN-γ and (E) IL-1β. BAL Fluid detects (F) IL-1β, (G) TNF-α and (H) IL-6 and protein cytokine levels by ELISA. mRNA changes are expressed as relative fold changes, and cytokine levels are expressed as pg/mL BALF. Data are expressed as mean ± SEM (n = 4-6). **, ##p <0.001 and *p <0.05 are through one-way analysis of variance. ## means significant compared to NC, ** means significant compared to DC.
Analysis of the BAL fluid secreted by cytokine proteins showed that after Ova challenge, the BALF levels of IL-1β (Figure 6F), tumor necrosis factor (TNF)-α (Figure 6G) and IL-6 (Figure 6H) were significantly increased ( DC and NC, p<0.001). SPAB (DC vs SPAB 34-306, p<0.001) or DM (DC vs DM, p<0.001) treatment significantly eliminated Ova-induced IL-1β, TNF-α and IL-6 secretion (6F -H).
We next tested whether SPAB can modulate the biochemical parameters (GPX, CAT, GSH, GSSG, SOD, MDA, and EPO) of oxidative stress induced by allergens in allergic lungs. Detect the activity of GPX (DC vs. NC, p<0.05) (Figure 7A), CAT (DC vs. NC, p<0.05) (Figure 7B) and SOD (DC vs. NC, p<0.001) (Figure 7C) in DC Enzyme activation is significantly reduced when attacked by allergens. Treatment with three different doses of SPAB restored the activity of GPX (DC vs. SPAB-306, p<0.05) (Figure 7A) and CAT (DC vs. SPAB-306, p<0.05) (Figure 7B) at the highest dose. Similar observations were made in the DM treatment group (7A-C). Figure 7 SPAB regulates oxidant/antioxidant levels in the whole lung and BALF of allergic mice. Homogenize the entire lung and measure markers of oxidative stress. (A) GPX activity, (B) CAT activity, (C) SOD activity, (D) GSH level, (E) GSSG level, (F) MDA level and (G) EPO/MPO activity in the whole lung and (H) MPO/EPO activity in BAL fluid. Data are expressed as mean ± SEM (n = 4-6). **, ##p <0.001 and *p <0.05 through one-way analysis of variance. ## means significant compared to NC, ** means significant compared to DC.
Figure 7 SPAB regulates oxidant/antioxidant levels in the whole lung and BALF of allergic mice. Homogenize the entire lung and measure markers of oxidative stress. (A) GPX activity, (B) CAT activity, (C) SOD activity, (D) GSH level, (E) GSSG level, (F) MDA level and (G) EPO/MPO activity in the whole lung and (H) MPO/EPO activity in BAL fluid. Data are expressed as mean ± SEM (n = 4-6). **, ##p <0.001 and *p <0.05 through one-way analysis of variance. ## means significant compared to NC, ** means significant compared to DC.
The levels of GSH and GSSG indicated that allergens caused a decrease in GSH levels in DC (DC vs. NC, p<0.001) (Figure 7D) and a simultaneous increase in GSSG levels (DC vs. NC, p<0.001) (Figure 7E). SPAB treatment restored the GSH level and the highest dose (DC vs. SPAB-306, p<0.05) (Figure 7D). In contrast, GSSG levels fell to normal levels at all three SPAB doses (DC vs. SPAB-34, -102, and -306, p<0.001) (Figure 7E). The MDA estimate showed that its level increased significantly after Ova attack (DC vs. NC, p<0.001) (Figure 7F). However, whether it was treated with DM (DC vs. DM, p<0.001) or SPAB (DC vs. SPAB-34, -102 and -306, p<0.001) (Figure 7F), the level was significantly reduced to normal.
Tests of peroxidase activity in the whole lung and BAL showed that MPO/EPO increased significantly in DCs challenged by Ova allergens (DC vs. NC, p<0.001) (Figure 7G and H). SPAB dose-dependently reduced allergen-induced MPO/EPO activity in the lungs (DC vs. SPAB-102 and -306, p<0.001) (Figure 7G) and BAL fluid (DC vs. SPAB-) at moderate and high doses. 102 and -306), p<0.05) (Figure 7H).
In order to summarize the in vivo anti-inflammatory response of SPAB, the human bronchial epithelial cell line BEAS2B was first treated with SPAB to test cell safety (Figure 8A). The results showed that treatment of BEAS2B cells with different doses of SPAB for 48 hours resulted in 20–25% cell death, up to 30 µg/mL SPAB. At 100 µg/mL, 37% of cell deaths were noticed. Therefore, 30 µg/mL is considered cell safe. Figure 8 SPAB inhibits the pro-inflammatory response of BEAS2B cells and HEK Blue cells in vitro. Incubate BEAS2B cells with different concentrations of SPAB for 24 hours, and measure (A) cell safety, expressed as a percentage of cell viability. BEAS2B cells were pretreated with SPAB for 24 hours, and then LPS SPAB was co-treated for another 24 hours. The secretion of (B) IL-6 and (C) TNF-α cytokine of the spent cell culture medium was measured by ELISA. AP-1/NF-kB-SEAP reporter gene detection. (D) AP-1/NF-kB-SEAP reporter gene HEK Blue cells were pretreated with different doses of SPAB for 6 hours, and then treated with TNF-α for 24 hours, and SEAP activity was measured. Data are expressed as mean ± SEM (n = 3). **, ##p <0.001 and *p <0.05 are through one-way analysis of variance. ## means significant compared with NC, ** means significant compared with LPS.
Figure 8 SPAB inhibits the pro-inflammatory response of BEAS2B cells and HEK Blue cells in vitro. Incubate BEAS2B cells with different concentrations of SPAB for 24 hours, and measure (A) cell safety, expressed as a percentage of cell viability. BEAS2B cells were pretreated with SPAB for 24 hours, and then LPS SPAB was co-treated for another 24 hours. The secretion of (B) IL-6 and (C) TNF-α cytokine of the spent cell culture medium was measured by ELISA. AP-1/NF-kB-SEAP reporter gene detection. (D) AP-1/NF-kB-SEAP reporter gene HEK Blue cells were pretreated with different doses of SPAB for 6 hours, and then treated with TNF-α for 24 hours, and SEAP activity was measured. Data are expressed as mean ± SEM (n = 3). **, ##p <0.001 and *p <0.05 are through one-way analysis of variance. ## means significant compared with NC, ** means significant compared with LPS.
Compared with NC, the LPS attack on bronchial epithelial cells induced the secretion of IL-6 (p<0.001) (Figure 8B) and TNF-α (p<0.001) (Figure 8C) cytokine proteins in the cell culture supernatant Significant increase. Pretreatment of cells with SPAB eliminated IL-6 (SPAB, 3 µg/mL-30 µg/mL, p<0.001) in a dose-dependent manner (Figure 8B) secretin and inhibited TNF- α (SPAB, 1 µg/mL-30 µg/mL, p<0.001) (Figure 8C) is released.
Next, in order to determine the molecular pathways involved in SPAB-mediated inhibition of pro-inflammatory cytokine levels, HEK-Blue TNF-α reporter cells were used. Treatment of HEK-Blue cells with TNF-α significantly induced NF-kB/AP-1 transcriptional activation 5.5-fold (p<0.001) (Figure 8D). In contrast, pretreatment of cells with SPAB moderately inhibited TNF-α-induced transcriptional activation (SPAB, 3 µg/mL – 10 µg/mL, 22 – 25%) (p<0.001) (Figure 8D). In general, the results of in vitro studies indicate that the NF-kB/AP-1 pathway may be involved in the inhibition of SPAB-mediated pro-inflammatory response. Obviously, the contribution of NF-kB signaling to the pathological improvement of SPAB-mediated asthma is limited. Therefore, the efficacy of SPAB in relieving asthma-related inflammation may also play a role in other ways.
The use of traditional medicine is becoming a new paradigm for the treatment of respiratory diseases. 37 Asthma is a chronic airway disease with limited treatment options. Using traditional medicine as a supplement to asthma treatment can help some asthma patients. In this study, we successfully evaluated the anti-asthma, anti-inflammatory and antioxidant properties of Sahastraputi-Abhrak-Bhasma (SPAB), a traditional Indian mica/biotite calcium-based drug. We have shown that in the lungs, SPAB can improve Ova allergen-induced inflammatory cell accumulation, peribronchial collagen deposition, mucus hypersecretion, epithelial thinking and allergen-induced oxidative stress in vivo; and in vitro pro-inflammatory cytokine expression and secretion. In addition, chemical analysis confirmed the presence of various therapeutically valuable silica-rich compounds in SPAB. The use of traditional medicines based on bhasma to treat asthma has been well studied. 23,38
Long-term use of the most popular corticosteroids and bronchodilators as treatments to reduce the symptoms of asthma can bring long-lasting side effects. 5 These side effects include immunosuppression and susceptibility to comorbidities and lung infections. 39 Therefore, alternative asthma treatments can reduce asthma symptoms without the need for immunosuppression. The use of traditional herbal medicines and 40 metal-based drugs8 to treat various respiratory diseases including asthma and chronic obstructive pulmonary disease is widely used as supplementary medicine. Bhasma is an ancient nanomedicine that has undergone biological treatment. 9 SPAB is a medicine based on mica (biotite)-calcium. When used directly, unprocessed mica minerals may have harmful effects on the human body due to the large amount of trace elements. 41 SPAB is heat-treated and chemically treated, and burned one thousand times to purify, detoxify and reduce particle size. In this study, in our mouse disease model, the use of SPAB significantly eliminated various human end points of asthma, indicating the therapeutic potential of SPAB and experimental verification.
Allergen-induced leukocyte infiltration into the lungs, especially eosinophils and lymphocytes, excessive mucus secretion, airway remodeling and subepithelial fibrosis lead to the morbidity and mortality of asthma. 3,42,43 Previous reports roughly indicated that abhrak bhasma has anti-inflammatory properties and provides relief from chronic cough and asthma. 44 In this study, oral treatment of allergic mice with SPAB can avoid the aforementioned asthma endpoints, indicating that SPAB has the potential to improve asthma-related airway inflammation. Consistent with our results, shatputi abhrak bhasma exerts immunomodulatory properties by stimulating the phagocytic activity of white blood cells. 18 In TB patients, shatputi abhrak bhasma treatment provides better results by regulating cough, fever, dyspnea, mucus, and hemoptysis. 45
Nanoparticle-based drugs are used to treat respiratory diseases. 37 SPAB’s chemical fingerprinting identified the presence of various compounds rich in silica, aluminum, iron, and magnesium. Rats fed with 2000 mg/kg Abhrak bhasma did not induce any DNA damage, nor did they show any signs of genotoxicity or cytotoxicity. 46 In addition, intravenous injection of Biotite mica did not induce any changes in tumor necrosis factor alpha (TNF) cytokine levels. -α), interleukin (IL)-6, IL-12 and interferon gamma (IFN-γ) indicate the immune safety of biotite. 47 Mice dietary supplemented with germanium biotite, which is rich in aluminum silicate, promotes lymphocyte proliferation and enhances the percentage of cytotoxic T lymphocytes (CTL). 48 Aluminum silicate exerts antiviral effects by reducing the titer of porcine reproductive and respiratory syndrome virus (PRRSV) in lung and lymphatic tissues, and may promote the clearance of PRRSV by increasing CTL. 48
In this study, whole lung and BAL analysis determined that Ova allergens induced cytokines, IL-5, IL-13, IL-33, IFN-ɣ and IL-1β, as well as IL-1β, TNF-α and IL- 6 Significantly eliminated after oral administration of SPAB. Th2 asthma is characterized by an elevated level of type 2 inflammation, which is mainly mediated by eosinophils, mast cells, T helper 2 cells, and basophils. 49 Increased levels of IL-5 are associated with increased eosinophilia. 50 In our study, the increase in IL-5 levels paralleled the increase in eosinophils. Th2 inflammation is also associated with increased IL-13 levels. 49 An increase in IL-13 levels induces hypersecretion of mucus in subepithelial fibrosis. 51 The allergen challenge in this study increased IL-13 levels and subsequent airway remodeling. IL-33 acts as an alarm during endothelial and epithelial cell damage. 52 In allergic inflammation, IL-33 plays a key role in type 2 innate immunity by activating mast cells, eosinophils, basophils, and macrophages. 52 IFN-ɣ plays an important role. 53 The use of blocking antibodies to treat asthma is limited because they are not sufficient to prevent allergic inflammation with complex equipment and severe asthma clinical problems. 50 Importantly, SPAB treatment significantly eliminated airway remodeling, eosinophilia, and related cytokine levels, indicating the therapeutic value of SPAB. Previous reports indicate that by increasing the expression of Th1 cytokines, IFN-γ and IL-12, biotite exerts antibacterial activity54 and reduces LPS-induced TNF-α levels. 55 On the contrary, dietary supplements of biotite can promote the expression of TNF-α, and IFN-γ is an anti-foot-and-mouth disease virus vaccine, which improves the immune stimulation ability of pigs and cattle by inducing humoral and cellular immune responses. 57 Our in vitro results are consistent with in vivo studies, indicating that SPAB reduces the secretion of cytokines in lung airway epithelial cells at least in part by regulating the transcriptional activity of NF-kB/AP-1. Part of the effect of SPAB on the transcriptional activity of NF-kB/AP-1 leaves room for the hypothesis that other pathways are involved in this process. In general, however, these results indicate that SPAB has anti-inflammatory and immunomodulatory properties.
Maghemite (γ-Fe2O3) has become a promising nano therapeutic material due to its biocompatibility, biodegradability and magnetic properties. 58 Previous reports have shown that γ-Fe2O3 has anti-inflammatory properties by inhibiting M1 polarization of macrophages and secretion induced by LPS. TNF-α, IL-1β and iNOS expression. 59 In rat alveolar macrophages, iron oxide inhibits the secretion of IL-6 by up-regulating the level of prostaglandin E2. 60 In osteoblasts, γ-Fe2O3 can induce integrin-α3 and α5 levels. 59 In osteoclasts, it inhibits the expression of MMP9 and cathepsin K. 59 Reduce the levels of TNF-α, IL-1β and iNOS, and increase the level of prostaglandin E2, indicating the possible role of maghamite in the current anti-inflammatory and anti-asthmatic properties of SPAB and SPAB.
Oxidative stress is an important confounding factor that aggravates the pathology of asthma61,62, and cellular non-catalytic mechanisms such as the glutathione system63 are the first line of defense to eliminate non-specifically produced free radicals under normal and pathological conditions. Catalytic antioxidant systems, such as SOD, peroxidase and catalase, in addition to act as signal intermediates that regulate cellular stress during asthma, they also target specific free radicals. 61 In the current work, SPAB can restore the levels of GPX, CAT, MPO/EPO, as well as GSH, GSSG and MDA. The antioxidant properties of abhrak bhasma were evaluated in rats using CCl4-induced hepatotoxicity and testicular hyperthermia64. 65 A single dose of abhrak bhasma can restore the levels of GSH64 and MDA66 in the liver and the ratio of GSH, GSSG, and GSH/GSSG in the testes. 65 In contrast, in the Drosophila system, SPAB can inhibit antioxidant components without any adverse effects on the general free radical scavenging ability. 67 Therefore, the ingredients present in SPAB can compensate for the decrease in antioxidant capacity. 67
The non-dose-dependent and non-linear pharmacological results of SPAB are the obvious limitations of this study, which may be due to two reasonable reasons. First, this may be due to the pleiotropic nature of SPAB that can act on several target molecules in living organisms. Drug receptor binding has proven to be an extremely complex process, and the extent of its functional output depends on several variables. Therefore, it is understandable to observe this type of dose-independent effect. 68,69 This effect is common in the context of in vivo studies, because the always observable effect depends on the physiological mediator environment of the inflammatory homeostasis. In fact, similar non-dose-dependent effects have been observed in various other herbs. 70 Secondly, the dose of SPAB may also cause several physiological response mechanisms, which may change its effective concentration or other innumerable factors. In order to maintain homeostasis by adjusting its role through the compensation mechanism. All in all, the specific purpose of conducting this study to verify the preventive anti-inflammatory effects and improvement potential of SPAB on asthma-related airway inflammation has been satisfactorily achieved. It should be admitted that, based on the observations of the current study, no conclusions can be drawn about the efficacy of SPAB. It is appropriate to conduct a separate detailed study in this regard, which will obviously become the most obvious derivative product in the near future.
The importance of traditional medicine as a treatment for asthma is undisputed. There is a need to determine safe, reliable and affordable treatment options as a holistic or complementary treatment for asthma management. We have verified the anti-inflammatory properties of the traditional Indian drug Sahastraputi-Abhrak-Bhasma (SPAB) through experiments, and proved that SPAB can reduce the typical symptoms of asthma-related airway inflammation, such as inflammation caused by allergens, excessive mucus secretion, and collagen Deposition and oxidative stress in vivo and secretion of cytokines in vitro. Therefore, this study provides strong enlightenment on the applicability of the biotite calcium-based drug Sahastraputi-Abhrak-Bhasma as an effective preventive adjuvant therapy for asthma management.
All data generated during the research period are included in the manuscript.
The author would like to thank Dr. LP Singh of the Central Institute of Architecture, Roorkee, India and his colleagues for their support of scanning electron microscopy, electron dispersive X-ray and XRD analysis; Dr. Vinamra Sharma and Mr. Pradeep Nain for their support of physical and chemical analysis; Ms. Deepika Mehra and Mr. Kamal Joshi Provide histopathology support; Mr. Vipin, Mr. Pushpender, Mr. Sonit, Dr. Sachin Sakat and Dr. GC Sar provided assistance in animal feeding, and Dr. Swati Haldar provided assistance in manuscript editing. We thank Mr. Brij Kishore, Ms. Priyanka Kandpal, Mr. Tarun Rajput, Mr. Gagan Kumar and Mr. Lalit Mohan for their prompt administrative support. Sahastraputi-abhrak-bhasma (SPAB) is a classic Ayurvedic medicine.
All authors have made significant contributions to the work of the report, whether in terms of concept, research design, execution, data acquisition, analysis and interpretation, or in all these areas; participating in drafting, revising or critically reviewing articles; final approval requirements Published version; agreed on the journal to which the article was submitted; and agreed to be responsible for all aspects of the work.
The author declares that there is no conflict of interest.
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