Deer Velvet Antler Improves Histopathological Changes of Allergic Airway Inflammation in Guinea pigs

Bronchial asthma is a chronic airway disorder. This serious health issue is present in 5 to 10 % people of all ages.1It is associated with repeated episodes of wheezing, shortness of breath, and coughing, especially at night or in the evening. These symptoms occur alone or in combination. Sometimes these symptoms are relieved spontaneously, but they usually require treatment. Asthmatic patients experience a great number of missed work and school days because of these symptoms.2

 The prevalence, morbidity, mortality, and economic burden associated with asthma have increased markedly throughout the world. 3According to the WHO, an estimated 300 million people of all ages are suffering from asthma, and approximately 250,000 people die from asthma every year.4  Asthma has been divided into two categories: Atopic asthma initiated by inhalation of an allergen, which leads to production of Ig-E antibodies and Non-atopic asthma occurs in individuals who are non-allergic and it is non Ig-E mediated.5

 Despite better understanding of asthma pathology and adherence to recommended treatments still many patients are experiencing uncontrolled asthma, these medicines especially steroids on chronic use develop serious side effects. Hence, it is is important to find alternative treatments.6,7

Given these facts,there is a need to develop a better and  more effective drug with minimum cost and adverse effects as asthma and allergy are life long diseases.

  For hundreds of years, people in China have utilized deer velvet antler that is a cartilaginous tissue without full calcification and being used as a medicinal herb and functional food to support general health maintenance8. Today, pharmacopeias from China, Korea, Japan, and Taiwan acknowledge velvet antler as a traditional medicine. Additionally, pharmacological activity related to immunomodulatory 9, anti-inflammatory 10 antiaging11, wound-healing12, and anticancer effects has been identified for velvet antler.13

 So far, 416 distinct proteins have been identified in DVA, for example, vimentin , β 3 subtype of hemoglobin, antimicrobial peptide, peptidoglycan recognition proteins, and pre-pro serum albumin.14 Regarding role of deer velvet antler in asthma, immunomodulatory effects of DVA have been reported in other studies. Aqueous extract of DVA has played a role in inhibiting T-cell proliferation, differentiation, and cytokine secretion after antigenic stimulation in vitro. It has also inhibited the release of IL-β1 and TNF-α from macrophages in mice.15

 It has inhibitory effects on the expression of many cytokines which are involved in pathogenesis of different TH2 mediated disease including asthma.16 it was  demonstrated in a study that prevention and alleviation of asthmatic symptoms after administration of DVA depend on elevation of Th1 and Treg cytokines and inhibition of Th2 and Th17 cytokines and Ig-E production and is able to regulate Th1/Th2 balance.17

 The role of DVA to protect and control allergic diseases like asthma has not been extensively studied. So, this study was designed to provide scientific basis for traditional use of DVA and to compare its anti inflammatory effects with dexamethasone.

Lung Histopathology

The lungs were dissected, the animals were killed under chloroform anesthesia, and they were fixed in a 10% formalin solution for 24 hours to create cassettes. To ascertain the lung morphology and level of inflammation, samples were cut into 5 µm slices after tissue processing, placed on glass slides, and stained with eosin and hematoxylin. Lung inflamation parameters (alveoler septal infiltrates, perivascular inflitrates and combined bronchus associated lymphoid tissue hyperplasia and peribrochial

inflitrates) were scored  as follows.

Score 0, for no inflamation

Score 1, for occasional cuffing with inflamatory cells (minimal)

Score 2, when most bronchus or vessels were surrounded by thin layer of 1-2  inflamatory cell layer (mild)

Score 3,when most bronchus or vessels were surrounded by thin layer of 3-4  inflamatory cell layer (moderate)

Score 4,when most bronchus or vessels were surrounded by thick layer more than 5  inflamatory  cell layers (severe)

The total lung inflammation was calculated by the addition of alveolar septal, perivascular, combined bronchus associatedlymphoid tissue hyperplasia and peribronchial inflammation scores for each animal .²¹

Airway epithelial desquamation was scored according to the fallowing criteria

Score 0, normal intact airway epithelium.

Score 1, elongation and distortion of the cuboidal and columnar epithelial cells lining the airways.

Score 2, elongation associated with infolding of the epithelium and narrowing of airway lumen.

Score 3, loss of epithelial cells resulting in broken airways.²²

Data Analysis: 

SPSS 20 was used to enter and evaluate the data. The histology scores for epithelial alterations and inflammatory states were recorded, together with their frequencies and probabilities. Moreover, the results were compared between groups using the Kruskal-Wallis analysis of variance (ANOVA). The post hoc analysis was conducted using the Mann-Whitney U test.

Table 2 presents the histological scores of lung inflammation and epithelial alterations in guinea pigs across different study groups. In negative group, no signs of perivascular, peribronchial, or alveolar septal infiltrates, nor bronchial epithelial changes, were observed. In contrast, the positive group exhibited moderate to severe perivascular, peribronchial, and alveolar septal infiltrates, along with epithelial alterations such as ciliated cell damage, mucous goblet cell hyperplasia, and/or squamous metaplasia. The DVA treated group showed these changes at minimal to moderate levels, while in the  dexa treated group, they ranged from minimal to mild.

Table 2: Histological parameters analyzed for NC, PC, DVA and DEXA treated groups.

 Figure 2a. Photomicrographs of (H & E stain, 400×). stained lung tissue samples from  NC, PC, DVA and DEXA treated groups for assessment of lung inflammation in (a) bronchial, (b) peri bronchial, (c) alveolar septal, and (d) perivascular inflammation .

 Fig 2b:Photomicrographs of (H & E stain, 400×). stained lung tissue samples from  NC, PC, DVA and DEXA treated groups for assessment of lung inflammation in perivascular area.

Overall histological scores for total lung inflammation and epithelial damage and narrowing were generated by taking means of individual lesion scores for each guinea pig of groups PC, DVA and DEXA respectively (Table 3a). NC group demonstrated no inflammation normal bronchial epithelium architecture (lesion score =0) 

Table 3 a: Effect of deer velvet antler and dexametasone on total lung inflammation of ovalbumin sensitized guinea pigs (n=6).

NC= negative control group, PC= positive control group, DVA=deer velvet antler and DEXA= dexamethasone treatment group.

Table 3b: Effect of deer velvet antler and dexametasone on bronchial epithelial damage of ovalbumin sensitized guinea pigs (n=6).

NC= negative control group, PC= positive control group, DVA=deer velvet antler and DEXA= dexamethasone treatment group.

When comparison of histological parameters was performed among groups by using Kruskal Wallis ANOVA, it was found that the difference was significant for total lung inflammation and bronchial epithelial damage and narrowing, all with p values 0.000 and 0.001(Table 4).

Table 4: Comparison of histological parameters in four study groups.

Fig 3 a: Effect of deer velvet antler and dexamethasone on total lung inflammation (Mean±SE) of ovalbumin sensitized guinea pigs (n=6). NC group demonstrated no  inflammation (lesion score =0).

PC= positive control group, DVA= deer velvet antler and   DEXA= dexamethasone treatment groups.

* p value ≤ 0.01, + p value ≤ 0.01 vs PC and #p value ≤ 0.01 vs PC

Fig 3 b: Effect of deer velvet antler and dexamethasone on epithelial damage and narrowing (Mean±SE) of ovalbumin sensitized guinea pigs (n=6). NC group demonstrated  normal bronchial epithelium architecture (lesion score =0).

PC= positive control group, DVA= deer velvet antler and DEXA=   dexamethasone treatment groups.

* p value ≤ 0.001, + Not Significant difference vs PC and # p value ≤ 0.05 vs PC

When pair wise comparison was evaluated for total lung inflammation by using Mann Whitney U Test, it was observed that group PC, DVA and DEXA had significantly increased total lung inflammation as compared to group NC with p values 0.002 in each case. DVA treated group had shown reduced total lung inflammation when compared with PC group with p value 0.004. Whereas DEXA treated group had significantly low inflammation as compared to PC group with p value 0.003. DVA treated group showed less reduction in inflammation score as compared to

DEXA group but difference was insignificant with p value 0.08. (Table 15)

When pair wise comparison was evaluated for airway epithelial damage and narrowing by using Mann Whitney U test, it was observed that group PC, DVA and DEXA had significantly enhanced airway epithelial damage and narrowing as compared to NC with p values 0.001, 0.002 and 0.006 respectively. DVA treated group had insignificantly reduced airway epithelial damage and narrowing when compared with PC group with p value 0.338. DEXA treated group reduced airway epithelial damage and narrowing when compared with PC group with p value 0.007. DVA treated group showed less reduction in airway epithelial damage and narrowing as compared to DEXA group but difference was insignificant with p value 0.067. (Table 5)

Table 5: Pair wise comparison of histological parameters in four study groups.

*** p value ≤ 0.001,** p value ≤ 0.01 and*p-value ≤ 0.05.

Because conventional asthma treatments often have side effects and may not be effective for many patients, there is increasing interest in safe, affordable, and scientifically validated herbal alternatives.23 An experimental model of induced asthma was established in guinea pigs to evaluate the beneficial effects of DVA on airway inflammation. Histological examination of lung tissue revealed that DVA treatment produced an improvement in lung inflammation comparable to that of dexamethasone, although the reduction in epithelial damage was less pronounced.

The study included four groups, with a normal control group serving as the reference. The other three groups were sensitized using ovalbumin (OVA) and alum. Among these, the OVA group received distilled water as a placebo, the drug control group was administered the standard drug dexamethasone for comparison, and the experimental group was treated with DVA. Sensitization with OVA led to marked infiltration of inflammatory cells and epithelial alterations in the airway mucosa, a well-established method for inducing airway inflammation.24

In this study, histological analysis of lung tissues revealed that inflammatory cells infiltration and airway epithelial desquamation was significantly increased in the sensitized, non treated group (PC) when compared with NC with p values 0.01 and 0.001 respectively. These histopathological parameters were absent in the NC. These observations are consistent with previous study that have shown increased number of inflammatory cells in the lung tissue and enhanced bronchial epithelial destruction after sensitization and challenge with ovalbumin in murine model of allergic asthma,25 these observations are considered as a sign of successful sensitization.

Administration of DVA to guinea pigs markedly ameliorated airway inflammation, inflammatory cells were reduced in lung tissues compared with the PC group with p value 0.01 but bronchial epithelial injury was reduced to lesser extent with p value 0.33.

Kuo et al., 17 had shown that DVA administration in asthmatic murine model significantly reduced infiltration of inflammatory cells in the lung connective tissue with p value 0.001,this statistical difference with the present study is may be due to differences in duration of treatment.

Yang et al., studied the protective effects of herba houttuyniae aqueous extract against ova‐induced inflammation in asthmatic mice and observed that airway inflammation scores were declined as compared to sensitized group with p value 0.05.27

In contrast to previously published studies that demonstrated statistically significant reductions in total lung inflammation (p < 0.01), the findings of the present study did not reveal a significant difference between DVA and dexamethasone treatment groups.28,29

Different studies proposed multiple possible anti-inflammatory mechanisms of DVA as mentioned in the literature review. Deer velvet antler has been used for decades in the treatment of various diseases, but there is still no definitive information about the active biochemical component that performs the main role in various pharmacological activities. In one study, 3.2 kDa polypeptide was isolated from DVA extracts, and in vitro analysis determined that this component enhanced natural killer cell activity, and cell-mediated and humoral immunity. This polypeptide promoted the secretion of Th1 cytokines like IL-2, TNF-α, IL-12 and IFN-γ, and decreased the secretion of Th2 cytokines (IL-4 and IL-10). This polypeptide could be useful for TH2 mediated diseases like multiple allergies including asthma.16

In conclusion, both DVA and dexamethasone demonstrated comparable effects on total lung inflammation; however, DVA showed no significant impact on epithelial alterations. These findings suggest that DVA may serve as a potential therapeutic agent for reducing lung inflammation in experimental asthma models.

DVA demonstrated an improvement in lung inflammation in an experimental model of allergic airway inflammation, showing effects comparable to those of dexamethasone. However, its ameliorative impact on alveolar and bronchial epithelial cells was less pronounced than that observed with dexamethasone. These findings suggest that DVA possesses promising anti-inflammatory potential for asthma management, warranting further investigation.

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