Background
Dermatophytes are pathogenic fungi that can invade keratinized structures and infect skin, hair, and nails of humans and other animals [
1]. In animal dermatomycosis, the most common pathogens are
Trichophyton mentagrophytes [
2],
Microsporum gypseum and
Microsporum canis [
3,
4]. The species
M. canis is found in humans and other animals; notably,
M. canis is zoonotic in nature.
M. canis is also known as one of the causes of dermatophytosis in rabbits [
5,
6]. A total of 21 isolates of
M. canis have been collected from rabbits with or without skin lesions [
1].
Rabbit dermatomycosis is a kind of highly infectious zoonotic contact dermatitis. The disease mainly causes dandruff, hair removal, exudation, crusting, folliculitis, and itching [
7]; This disease can also result in rabbit malnutrition, growth retardation, feed remuneration reduction and even death. Furthermore, dermatomycosis directly affects the quality of skin, reproductive performance, and survival rate of young rabbits. In many warrens, dermatomycosis occurs at an incidence rate of 30 % to 100 %, pup growth rate decreases by 20 % to 30 % and mortality rate ranges from 20 % to 40 % before weaning [
8].
Dermatophytosis is treated by using various antifungal agents, such as clotrimazole, terbinafine, and ketoconazole [
9]. However, drug resistance, toxicity, and drug-drug interactions limit the use of these treatments [
10,
11]. Medicinal plants play an essential role in Chinese ethnoveterinary medicine [
12] because these plants can effectively treat various ailments [
13]. Approximately 40 % of the total medicinal consumption in China is attributed to traditional medicines [
12]. Antimicrobial, fungicidal, and antioxidant properties of many therapeutic plant extracts have been widely reported [
14]. These medicinal properties are caused by active chemical constituents in their roots, stems, leaves, seeds, and bark.
The bark of a
Phellodendron tree has been used in traditional Chinese medicine for thousands of years.
P. amurense is commonly used to treat gastroenteritis, abdominal pain or diarrhea, and various inflammatory diseases, including arthritis and dermatophytosis. The main bioactive components of
P. amurense are berberine hydrochloride and palmatine hydrochloride [
15]. Previous studies have implied a number of biological activities of berberine, including anti-secretory, anti-inflammatory, anti-bacterial, anti-malarial, anti-mycobacterial [
16], anti-tumor and anti-cholesterol activities. Berberine and palmatine were found inhibited CYP1A1.1- and CYP1B1.1-catalyzed 7-ethoxyresorufin O-deethylation (EROD) activities. Kinetic analysis revealed that berberine noncompetitively inhibited EROD activities of CYP1A1.1 and CYP1B1.1, whereas palmatine and jatrorrhizine caused either competitive or mixed type of inhibition [
17]. In previous study, berberine and palmatine were screened to determine their inhibitory activities various dermatophytes [
18]; results revealed that berberine exhibited activity against
M. canis (MICs, μg/mL >1000). To determine the antifungal mechanism of
Phellodendron amurense against
M. canis, we used berberine hydrochloride, palmatine hydrochloride and combined treatment
in vitro and vivo experiments. Our results could provide a scientific basis for the treatment of skin diseases with natural drugs.
Discussion
Owing to zoonotic transmission of
M. canis, efforts for effective treatment are necessary. Various parts of traditional medicinal plants were reportedly used against dermatophytosis.
Phellodendron, a deciduous tree species widely grown in China, is a medicinal plant commonly used to treat various ailments, including gastroenteritis, abdominal pain, diarrhea, abscess, and other inflammations or swellings [
29]. Its antibacterial activity against
Staphylococcus aureus,
S. albus, α-streptococcus, β-streptococcus,
Proteus and
Bacillus Dysenteriae, as well as antifungal activity on
Cryptococcus neoformans and
Trichophyton purpureatum, have also been reported [
30]. Although reports on the effecacy of medicinal plants against fungi have been presented, studies on their active mechanism are very few. Wang [
31] investigated the antifungal mechanisms of cinnamon oil and pogostemon oil complexes on intestinal
Candida infections. However, data are limited to microscopic evaluation using a scanning electron microscopy (SEM) and TEM.
Berberine hydrochloride and palmatine hydrochloride are the main bioactive components of
Cortex phellodendri, with quantities of around 0.6 % and 0.3 %, respectively [
18]. Berberine and palmatine, are procured from roots of
B. aristata, B. petiolaris, B vulgaris, B.aquifolium, B. thumbergii, B. asiatica and among Chinese herbs it’s primary sources are
B.sargentiana, Phellodendron amurense and
Coptis chinensis from rhizomes and bark respectively. Both are members of a group of alkaloids that have been reported to display various biological and pharmacological activities. Some experiments showed that berberine significantly attenuated
C. pneumoniae infection-induced VSMC migration (
P <0.05) and berberine suppressed the protein expressions of MMP3 and MMP9 caused by
C. pneumoniae infection in a dose-dependent manner (
P <0.05).
C. pneumoniae infection-induced increase in the phosphorylation level of Akt at Ser473 was inhibited by the treatment with berberine (
P <0.05). Taken together, those data suggestted that berberine inhibits
C. pneumoniae infection-induced VSMC migration by downregulating the expressions of MMP3 and MMP9 via PI3K [
32]. Park [
33] reported that berberine can inhibit
Candida krusei with MIC < 1 μg/ml and palmatine can inhibited
Candida tropicalis with MIC of 16 μg/ml. Zeng [
34] found the MIC of berberine to be 640 μg/ml after 16 h incubation based on colony counting of
S. aureus 2 × 10
7 CFU/ml. Volleková [
20] tested berberine, palmatine and jatrorrhizine for their inhibitory activity against a variety of dermatophytes and two
Candida species and found that berberine and palmatine exhibit certain antifungal activity (MIC 500 to ≥ 1000 μg/mL). In our study, the MIC of berberine and palmatine was 1 mg/mL and the colony count of
M. canis was 1.0 × 10
6 CFU/mL less than 2 × 10
7 CFU/ml reported by Zeng [
34], the colony number and microbes kinds were different might lead to a different MIC. In this study, MIC of clotrimazole was determined to be 15 μg/ml, which is higher than previously reported (0.06 ~ 0.125 μg/ml) [
35] and much closer with the results 0.5 ~ 8 μg/ml [
36]. We found that the MIC of clotrimazole was lower than the tested monomers. Thus, clotrimazole, which is a very effective antifungal drug, should have higher fungicidal activity. Interestingly, the present study showed that the initial effect of clotrimazole was lower than that of the monomers or their combination (Fig.
12). This result may be caused by the immunosuppressant cyclophosphamide used in the experiments [
37], indicating that chemical agents can limit the bioactivity of clotrimazole; as a result, lower efficacy is obtained. Compared with synthetic antifungal medications, natural drugs could evade this problem and may be used safely and effectively.
Electron microscopy showed that certain concentration of monomers could have damaging effects on cell membrane, nucleus and organelles of M. canis cells after 18 and 30 h of incubation; thus, the normal growth of the fungi was inhibited. Antifungal mechanisms of monomers possibly rely on this specific effect on fungal cell wall and cell membrane.
Other antifungal mechanisms of drugs were mainly observed on the basis of the following aspects: (1) genetic material quantitative analysis using a laser scanning confocal microscope and scanning image cells [
38]; (2) analysis of cell nucleic acid and protein drug effects on fungal DNA synthesis cycle [
39]; (3) cell energy metabolism and other effects [
40] .
P-GAL4 gene belongs to the GAL4 family, containing only one GAL4 domain, which is specific to fungal species. The protein encoded by GAL4 has different functions in different microorganisms. Hon [
41] reported that it can accelerate the synthesis of heme in
Saccharomyces cerevisiae. Masloff [
42] reported that it could regulate fungal reproduction. GAL4 family proteins are also involved in nitrogen metabolism, particularly under medical stress;
M. canis could increase its metabolism activity to reduce damage and overcome the influence of antifungal drugs. After 6 h of incubation with palmatine and clotrimazole, P-GAL4 expression was up-regulated significantly compared with that in other groups (
P <0.05). After 18 and 30 h, the expression of P-GAL4 gene in B-P treatment group was also up-regulated significantly compared with other groups (
P <0.05) (Fig.
3). These results suggested that the antifungal activity of the B-P combination may occur slowly but is more effective than clotrimazole alone. Palmatine possibly up-regulate P-GAL4 activity earlier than berberine. This result is similar to that of Zeng
et al. [
34], in which the antimicrobial activity of berberine was observed after 16 h of incubation.
The protein encoded by FSH1 is a serine hydrolase containing a Ser ⁄ His ⁄ Asp active site. This protein is an esterase that can hydrolyse phosphate ester compounded by analysing its active site and other protein structures [
43]. It belongs to a large, multifunctional ab-hydrolase subfamily, the FSH family [
44]. As a proteinase, FSH can degrade proteins to amino acids and oligopeptides; as the amidase of an endogenous signal factor, FSH can regulate metabolism. We found that palmatine could up-regulate the expression of FSH1 in as early as 6 h of incubation. After 18 and 30 h, the antifungal activity of B-P combination was evident, and FSH1 expression was significantly upregulated (Fig.
4). The function of FSH1 could be the same as that of P-GAL4.
The PQ-LRP protein is a membrane-binding protein containing a pair of ring structures. As a secondary signal, the second ring is quite important to cysteine vector localization in the lysosome [
45] and it is useful for the anabolic metabolism of cysteine. Fedorova [
46] identified the PQ-LRP gene when they analyzed the genomic DNA of
Aspergillus fumigatus; they proposed that the PQ-LRP protein could be a general component of fungal cells and thus, related to fungal growth and development. PQ-LRP gene up-regulation could enhance cysteine metabolism; PQ-LRP protein could produce cysteine by decomposing disulfide bond of proteins, which increase the pathogenicity of
M. canis. After 6 h of incubation, PQ-LRP expressions were up-regulated in all of the drug-treated groups compared with NC control. After 18 h, PQ-LRP expressions in B and B-P groups were up-regulated compared with those in the NC group. After 30 h, PQ-LRP expressions in B-P group was significant higher than that in other groups except the P group (Fig.
5).
NADH1, which is encoded in the mitochondria, is an important part of the respiratory chain. NADH1 passes electrons in the respiratory chain of mitochondrial oxidation to harness energy through ATP. B-P combination enhanced the expression of NADH1 mainly after 18 h of incubation (Fig.
6). The results of NADH at the enzyme level were slightly different from that obtained from gene expression. However, after 30 h, the results were almost same. P, B-P combination, and NC were significantly higher than the other groups (
P <0.05) based on real-time PCR. This result was also observed at the enzyme level (Fig.
11).
Ribonucleoproteins (RNPs) are RNA-associated proteins that combine with mRNA and non-coding RNAs to form RNP complexes. In the context of structured RNPs, the activity and stability of many RNAs are regulated post-transcriptionally. The composition of an RNP dictates RNA fate, reflecting aberrations that subject the RNA to quality-control degradation pathways. After 6 h to 18 h of incubation, no significant difference in terms of RNP expression was found between the groups, which means that the influence of drugs on RNP expression were minute. After 30 h of incubation with the clotrimazole and combined group, the mRNA expression of RNP was significantly higher than that of the other groups (
P <0.05) (Fig.
7).
Xylose reductase is an intracellular enzyme commonly found in yeast and filamentous fungi. This enzyme occurs in the cytoplasm of microorganisms, where it catalyzes the first step of xylose metabolism by reducing xylose to xylitol with the concomitant oxidation of NAD(P)H to NAD(P) + [
47]. After 6 h of incubation, the mRNA expression of NDR in P and B-P group were significantly higher than that of the other groups except for the NC group. After 18 h of incubation, the mRNA expression of NDR in the B-P group was significantly higher than the other groups (
P <0.05) (Fig.
8), which is similar with the results of NADH1 real time PCR after 18 h.
Chitinases are enzymes that cleave the β-(1,4) glycosidic bond of chitin, a structural component in fungal cell walls. The carbohydrate-active enzyme (CAZy) glycoside hydrolase family 18 (GH18), as defined by amino acid sequence similarity [
48], contains a large number of chitinases expressed in prokaryotes and eukaryotes. It is subdivided into two subfamilies, bacterial-type and plant-type family 18 chitinases, according to sequence similarity, active site construction, preferred activity (exo-chitinases versus endochitinases, respectively), and occurrence in different organisms. It has been hypothesized that bacterial-type chitinases in fungi and bacteria are used to process chitin as a carbohydrate [
46]. After 30 h of incubation, the mRNA expressions of SC in PC and B-P groups were significantly higher than those in the other groups (
P <0.05) (Fig.
9).
Zinc transporter zupT (ZTZ) mediates zinc uptake. It transport zinc from within the cell, either out of the cell across the plasma membrane or into intracellular compartments, which reduces cytosolic concentrations [
49]. After 30 h of incubation, the mRNA expressions of ZTZ in PC and B-P groups were significantly higher than those in the other groups (
P <0.05) (Fig.
10).
Real-time PCR results showed that palmatine likely induced the up-regulation of certain gene expressions earlier than berberine. By contrast, berberine possibly regulated antifungal activity, especially after 18 h, in which B-P shortened the life cycle of M. canis and eradicated this fungus. Clotrimazole could also increase the mRNA expressions of PQ-LRP, PGAL4 and FSH1 for a short period but failed to maintain such high expression.
In vivo study, it was observed that the lesions in the B-P treatment groups started to recover from day 7. No significant differences between P, B and B-P treatment groups were found, while average score of B-P group was lower than P, B group from day 7. Treatment with clotrimazole subsided skin lesions, but there was no significant difference when compared with P and B group but showed significant difference when compared with B-P group from day 7. The results of PAS were also consistent with the results of clinical experiment. Clotrimazole is well-documented antifungal agent, and the performance of B-P in this study was somewhat better than the performance of clotrimazole.
The monomers from P. amurense possibly exhibited antifungal activities by disrupting fungal cell wall and cell membrane and by increasing the expressions of energy metabolic genes; thus, the life cycle of M. canis was shortened. Our results showed that the mixture of palmatine hydrochloride and berberine hydrochloride could be used effectively to treat rabbit dermatomycosis caused by M. canis.
Authors’ contributions
XCW carried out the MIC and RT-PCR experiment; XCW, JQA and PLJ performed carried out the animal experiments and skin histology PAS staining experiments; WQ and LY carried out ultrastructural analysis experiment; BGL, XCW designed the experiments. All authors read and approved the final manuscript.