Traditional Chinese medicine suggests that renal deficiency is a causative factor of asthma, and tonifying kidney drugs are believed to be an appropriate and beneficial treatment. The adrenal medullary chromaffin cells (AMCC) transition to the neuronal phenotype is known to occur in asthma, as evidenced by degranulation of chromaffin granules, decline of epinephrine (EPI) and phenylethanolamine-n-methyl transferase (PNMT), and obvious alterations in cellular architecture. In this study, rats were sensitized and challenged with ovalbumin, then treated with Kidney-Tonifying Recipe (KTR) to evaluate the therapeutic effect. Tissues were evaluated for changes in pathology and EPI, PNMT, and peripherin expression. Degranulation of chromaffin granules and appearance of neurite-like process were found in AMCC from asthmatic rats, and these changes were corrected by KTR treatment. EPI and PNMT expressions were decreased in asthmatic rats and increased by KTR treatment. Peripherin expression was increased in asthmatic rats and decreased in the KTR-treated group. Morphological changes and decreases in EPI were observed when cultured AMCC were exposed to sera from asthmatic rats
Traditional Chinese medicine (TCM) considers renal deficiency as a principal cause of asthma. A tonification strategy for kidney is often used to treat asthma in TCM, and many consider this to be a highly effective therapeutic approach [
Asthma attacks are induced by the dysfunction of cholinergic and adrenergic nerves and their cognate receptors. Since there is a lack of adrenergic nerves innervating the human airway smooth muscles, the relaxation of airways is mainly regulated by epinephrine (EPI) binding to adrenergic receptors. Adrenal medullary chromaffin cells (AMCC) are the primary cells that secrete EPI [
The role and mechanisms underlying the beneficial effects that are observed upon use of tonifying kidney drugs are unclear. We hypothesized that tonifying kidney drugs affect the structure and function of AMCC. In this study, asthmatic rats were treated with a classic TCM formula known as Kidney-Tonifying Recipe (KTR), and the effects of the kidney-tonifying drugs on the alteration of structure and function of AMCC in asthma were investigated.
KTR is composed of
All animals used in this study were conventionally bred 6- to 8-week-old male Sprague-Dawley rats (Experimental Animal Center of Central South University, Changsha, China). This study was carried out in strict accordance with the recommendations from the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, USA. The protocol was approved by the Ethics Committee at the Asthma Research Institute, Hunan Province.
Twenty-four rats were divided into three groups using a random digits table (
After determination of bronchial responsiveness, blood from each rat was obtained by cardiac puncture, and the serum was inactivated by heating at 56°C for 30 minutes, then filtered through a 0.22
Medium A was prepared as a mixture of 85% DMEM serum-free medium (Gibco, USA) and 15% serum from experimental rats. According to the origin of serum, three Medium A subgroups were generated: serum from control rats (control serum), serum from asthmatic rats (asthma serum), and serum from KTR-treated rats (KTR serum). Medium B was prepared as a mixture of 15% fetal bovine serum (Gibco, USA) and 85% DMEM serum-free medium.
Adrenal glands were obtained from conventionally bred 6- to 8-week-old male Sprague-Dawley rats. Medullae were carefully freed from the capsular and cortical tissue under sterile conditions and incubated with Hanks’ balanced salt solution. Subsequently, medullae were dissociated with 0.1% type I collagenase dissolved in glucose-enriched phosphate-buffered saline for 40 minutes. After harvesting by centrifugation and resuspending in culture medium B, the cells were adjusted to a cell concentration of 5-6 × 106/mL and incubated at 37°C. Medium was changed after 24 hours, and then every 48 hours thereafter, to remove dead cells and cell debris.
The cultures in medium B were maintained for a total of 5 days and divided into the three experimental groups for treatment with one of the different medium A subgroups described above. Seventy-two hours after the AMCC cells were treated with medium A (experimental serum), the cells and the supernatants were collected for subsequent analysis.
The tissue of lung and adrenal medulla were fixed in 4% paraformaldehyde and then embedded in paraffin. Tissue sections (4
Adrenal medulla and AMCCs were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2. Three hours later, specimens were postfixed in buffered 1% OsO4 for 1 hour following by hydrating in a decreasing ethanol series and embedding in Epon-Araldite. Ultrathin sections (70 nm) from the different specimens were stained with uranyl acetate and lead citrate and examined with an H-7500 transmission electron microscope (Hitachi, Japan). Ultrastructural changes were assessed by pathologists who were blinded with respect to treatments.
The levels of EPI, corticosterone, and NGF in serum or cellular supernatant were quantified by commercially available ELISA kit and antibodies, according to the protocol provided by the supplier (R&D, USA). Immunoreactivity was determined by an ELISA reader at 450 nm.
Total RNA was extracted from tissues or cells using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. For RT-PCR reactions, primer sequences were designed that yielded 137 and 307 bp products of peripherin and GAPDH, respectively. The gene-specific primer sequences were as follows: 5′-GGTGGAGGTAGAGGCAACA-3′ (forward) and 5′-TCGGACAGGTCAGCGTATT-3′ (reverse) for peripherin, and 5′-TGAACGGGAAGCTCACTGG-3′ (forward) and 5′-TCCACCACCCTGTTGCTGTA-3′ (reverse) for GAPDH. The PCR amplification program consisted of preheating at 94°C for 5 minutes and 35 cycles of denaturing (94°C, 30 seconds), annealing (60°C, 30 seconds), and extension (72°C, 1 minute), followed by a final extension at 72°C for 10 minutes. The PCR products were electrophoresed on a 2% agarose gel to confirm size. Results were normalized to GAPDH, which was used as an internal control.
After embedding in paraffin at 4°C overnight, the tissues were sectioned (4
Total proteins were extracted by RIPA Lysis Buffer (Takara, Japan). Thirty micrograms of protein were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then electrotransferred to polyvinylidene fluoride (PVDF) membrane at 120 V for 1.5 hours. Nonspecific binding was blocked by incubating with 0.05 g/mL skim milk powder at room temperature (20°C) for 2 hours, and then membranes were incubated at 4°C overnight with rabbit anti-rat peripherin polyclonal antibodies (1 : 1000; Millipore, USA). After washing with PBST, the membranes were incubated at 37°C for 1 hour with secondary antibodies (1 : 5000; Sigma) and visualized by using the enhanced chemiluminescence reagent system (Pierce, USA). Results were normalized to GAPDH, which was used as an internal control.
Data are presented as mean ± standard deviation
Administration of increasing concentrations of histamine caused gradual increases in airway resistance in each group. When the concentration of histamine reached 0.16 mg/mL and above, airway resistance in the asthma group was higher than that in the control group (
Changes in rat airway resistance in response to histamine exposure. Rats in three groups were treated with aerosolized saline or increasing doses of histamine generated by an ultrasonic nebulizer. Airway resistance was measured by whole-body plethysmography and expressed as the index of percent increase in airway resistance as compared to the peak of the reaction with baseline. The values are expressed means ± SD (
Morphological changes in lung tissue were evaluated by examining lesions in asthmatic rats. We found that, compared with the control rats, asthmatic rats had an increased shedding of epithelial cells, remarkable infiltration of eosinophils, and lymphocytes surrounding the airway. These pathological changes were found to be reduced in the KTR-treated rats. The results showed that the airway wall thickness in asthmatic rats (
Pathological changes in rat lung tissue in response to induced asthma and KTR treatment. Representative micrographs (HE staining, 100x) of lungs from control rats (a), asthmatic rats (b), and KTR-treated asthmatic rats (c).
Compared with the control rats, asthmatic rats presented with vacuolar degeneration, tissue edema, and increased lipid in AMCC, as observed under light microscope. However, these lesions were reversed by KTR treatment (Figures
The pathological changes of rat adrenal medulla in response to induced asthma and KTR treatment. Representative micrographs (HE staining, 100x) of adrenal medulla in control rats (a), asthmatic rats (b), and KTR-treated asthmatic rats (c). Representative electron micrographs (10000x) of adrenal medulla in control rats (d), asthmatic rats (e), and KTR-treated asthmatic rats (f). The arrows show vacuolar degeneration (black arrow), chromaffin granules (red arrow), swollen cytoplasm, and mitochondria (blue and white arrows), respectively.
Electron microscopy revealed a regular morphology of AMCC in control rats, which contained a clear structure of mitochondria and abundant chromaffin granules. Lesions were evident in asthmatic rats, including swollen cytoplasm and mitochondria, and decreased chromaffin granules. These pathological changes were relieved in the KTR-treated rats (Figures
EPI levels in asthmatic rats were lower than in control rats (
Changes in levels of EPI, corticosterone, and NGF in rats in response to induced asthma and KTR treatment. The levels of EPI, corticosterone, and NGF in rats (
Under normal physiologic conditions, PNMT is primarily expressed in chromaffin cells and absent in neurons. We found that the immunostaining of PNMT was distributed throughout cytoplasm. In control rats, the density of immunostaining was remarkably high in the adrenal medulla. Compared with the control rats, the expression of PNMT in asthmatic rats was decreased (
Changes in PNMT and peripherin protein expression in adrenal medulla of rats in response to induced asthma and KTR treatment. The expressions of PNMT and peripherin in the adrenal medulla of rats were detected by immunohistochemistry. Representative images of immunostaining (DAB, 100x) for PNMT are presented for control rats (a), asthmatic rats (b), and KTR-treated asthmatic rats (c). Representative images of immunostaining (DAB, 100x) for peripherin are presented for control rats (e), asthmatic rats (f), and KTR-treated asthmatic rats (g). Bar graphs on the right panels represent the average optical density of PNMT (d) and peripherin (h). The values are presented as means ± SD (
Peripherin is rarely expressed in chromaffin cells and has high expression in neurons. We found that peripherin immunostaining was mainly localized in the cytoplasm. Compared with control rats, the density of immunostaining in asthmatic rats was higher than that in control rats (
Compared with control rats, asthmatic rats had significantly increased amounts of peripherin mRNA and protein expression (
Changes in peripherin gene and proteins expressions in rat adrenal medulla tissue and AMCC in response to induced asthma and KTR treatment. Total RNA and cellular protein were isolated from adrenal medulla tissue in rats (
Electron microscopy indicated that cultured chromaffin cells treated with control serum
Pathological changes of AMCC
Compared with cells treated with control serum, EPI levels were sharply decreased in the supernatant of cells treated with asthmatic serum (
Compared with the control cells, AMCC treated with asthmatic serum showed a significant increase in peripherin mRNA and protein expressions (
Kidney-tonifying drugs are often used to treat asthma in TCM. Previous reports have suggested that the effects of these drugs are mediated by their ability to upregulate plasma cortisol production by stimulating the adrenal cortex [
EPI is a
NGF is the most important neurotrophic factor involved in neuronal growth, survival, and differentiation. NGF expression and activity is enhanced in asthma [
During the development and regeneration of nerve cells, peripherin (a type III intermediate filament) plays an important role in cellular architecture through regulating axon formation, thus giving nerve cells their unique morphology [
PNMT, the rate-limiting enzyme that catalyzes nor-EPI to EPI, is mainly expressed in adrenergic chromaffin cells, rather than in nerve cells. Decreases in PNMT mediate the transition from AMCCs to neurons. Glucocorticoids are known to promote the expression of PNMT in the adrenal medulla [
In the current study, PNMT expression was found to be decreased in asthmatic rats, consistent with decreased EPI and increased NGF levels, and KTR treatment inhibited these changes. Despite increased corticosterone levels in asthmatic rats, there remained a trend for NGF-induced transformation of AMCC. The further increase in corticosterone in asthmatic rats treated by KTR may act as a compensatory mechanism, as supplementation with exogenous glucocorticoids is a highly effective treatment in asthma patients. To some extent, these data suggest that the effect of KTR in treating asthma is dependent on maintaining the phenotype of AMCC, which may be ascribed to correcting the imbalance of NGF and glucocorticoids; as a result, EPI concentrations would be expected to be increased, thus relieving bronchospasms.
To test this hypothesis, we cultured AMCC
In summary, it can be concluded that administration of KTR to asthmatic rats leads to upregulation of EPI and prevention of AMCC transformation to neurons, thus allowing for normal endocrine function.
This paper was supported by Grants from the National Natural Science Foundation of China (nos. 30801505, 30800502, and 81070026).