[Effect of butyl alcohol extract of Baitouweng Decoction on vaginal mucosal neutrophil chemotaxis in vulvovaginal candidiasis mice].

To investigate the effects of butyl alcohol extract of Baitouweng Decoction(BAEB) on neutrophil chemotaxis in vaginal mucosa of mice with vulvovaginal candidiasis(VVC). Seventy-two SPF female Kunming mice were randomly divided into normal control group, model group, fluconazole group, BAEB low-dose group, middle-dose group and high-dose group. Subcutaneous injection of estradiol benzoate was conducted to induce pseudo-estrus, and then 2×10~6 CFU·mL~(-1)of Candida albicans was inoculated into vaginal lumen, followed by drug treatment for 7 days. Gram staining was used to observe the morphological changes of C. albicans in vagina; vaginal fungal load was detected on agar plate. Histological changes of vaginal tissues in mice were observed by HE staining. Lactate dehydrogenase(LDH), interleukin-6(IL-6) and tumor necrosis factor(TNF-α) levels in mouse lavage fluid were detected by enzyme-linked immunosorbent assay(ELISA). Neutrophils in vaginal lavage fluid was observed and counted by using Pap smear. The levels of IL-8 and MIP-2 in vaginal mucosa were detected by ELISA. IL-8 and MIP-2 mRNA levels in vaginal mucosa of mice were detected by qRT-PCR. The results showed that as compared with the normal group, VVC model group had a large number of hyphae and a high level of fungal loadinvagina. The vaginal mucosa was completely destroyed, the number of neutrophils increased, and the protein and mRNA levels of IL-8 and MIP-2 were up-regulated. After BAEB treatment, the hyphae of the treatment group was decreased, the fungal load was decreased, the impaired mucosa showed different degrees of improvement, the inflammatory factors were decreased to varying degrees, and the protein and mRNA levels of chemokine IL-8 and MIP-2 were down-regulated. In conclusion, BAEB may be used to treat VVC by inhibiting vulvovaginal candidiasis via blocking neutrophils recruitment into vagina.

[Inhibitory effect of extract of Coptidis Rhizoma on invasion of Candida albicans hyphae in vitro].

The aim of this paper was to investigate the inhibitory effect of extract of Coptidis Rhizoma(ECR) on invasion of Candida albicans hyphae in vitro.XTT reduction method was used to evaluate the metabolic activity of C.albicans.The colony edge growth of C.albicans was observed by solid medium.The growth of C.albicans hyphae was determined on semi-solid medium.The morphology and viability changes of C.albicans hyphae were assessed by scanning electron microscope and fluorescence microscope.qRT-PCR method was used to detect the ALS3 and SSA1 expression of C.albicans invasin genes.The results showed that the metabolic viability by XTT method detected that the activity of C.albicans was gradually decreased under the intervention of 64,128 and 256 mg·L-1 of ECR respectively.128,256 mg·L-1 of ECR significantly inhibited colony folds and wrinkles on solid medium and the hyphal invasion in semi-solid medium.Scanning electron microscopy and fluorescence microscopy showed that 128,256 mg·L-1 of ECR could inhibit the formation of C.albicans hyphae.qRT-PCR results showed that the expression of invasin gene ALS3 and SSA1 was down-regulated,and especially 256 mg·L-1 of ECR could down-regulate the two genes expression by 4.8,1.68 times respectively.This study showed that ECR can affect the invasiveness of C.albicans by inhibiting the growth of hyphae and the expression of invasin.

Sodium houttuyfonate in vitro inhibits biofilm dispersion and expression of bdlA in Pseudomonas aeruginosa.

Biofilm dispersion is the last step in the development of biofilms, and allows bacteria to spawn novel biofilms in new locales. In the previous studies, we found that sodium houttuyfonate (SH) is effective at inhibiting biofilm formation and motility of Pseudomonas aeruginosa. Here, we investigated the effect of SH against the biofilm dispersion of P. aeruginosa by an in vitro model. The results show that the plant derivative, SH, could effectively inhibit both biofilm dispersion of P. aeruginosa, and gene and protein expression of the key biofilm regulator BdlA in a dose-dependent manner. Furthermore, our presented results suggest that SH can penetrate into the biofilm of P. aeruginosa to repress the biofilm life cycle. Therefore, these results indicate that the antimicrobial activity of SH may be partially due to its ability to disrupt biofilm dispersion in P. aeruginosa.

[Therapeutic potential of n-butanol extract of Pulsatilla decoction in a murine model of ulcerative colitis induced by DSS combined with Candida albicans colonization].

To investigate the mechanism of n-butanol extract of Pulsatilla decoction (BAEB) against murine ulcerative colitis (UC) model induced by DSS combined with Candida albicans (CA) colonization, mice were randomly divided into normal control group, DSS group, DSS+CA group, BAEB high, medium and low dose group, and positive drug Mesalazine group. The general condition of mice was observed, fungal loads of murine intestinal contents were detected by plate method, colonic pathological change of mice was examined by HE staining. ASCA in serum and IL-6, IL-8, IL-1ß, HBD-2, HBD-3 in colonic mucosa were detected by ELISA. The results showed that, compared with DSS group, the general condition and ASCA in serum had no obvious change for DSS+CA group, but the fungal loads in intestinal contents, the colonic pathological damage, and the levels of IL-6, IL-8, IL-1ß, HBD-2, HBD-3 in colonic mucosa were greater than that of DSS group. High dose of BAEB group and Mesalazine group could improve the colonic pathology, decrease IL-6, IL-8, IL-1ß, HBD-2, HBD-3 expression level. In conclusion, BAEB could effectively improve the UC symptoms in mice induced by DSS combined with CA colonization, and inhibit the inflammatory factors such as IL-6, imply that BAEB is of important value for the treatment of intestinal fungal-related colitis.

[Butyl alcohol extract of Baitouweng decoction inhibits Candida albicans cell membrane].

To study the inhibitory effect of butyl alcohol extract of Baitouweng decoction(BAEB) on Candida albicans cell membrane. The effects of BAEB on the activity of C. albicans were observed by Spot assay. The changes of intracellular osmotic pressure of C. albicans after BAEB intervention were detected by microtiter plate reader. The effect of BAEB on cell membrane permeability of C. albicans were observed by fluorescence microscopy. The content of ergosterol in C. albicans cell membrane was detected by high performance liquid chromatography, and the expression of ergosterol biosynthesis related genes in cell membrane was detected by qRT-PCR. The results showed that the activity of C. albicans was significantly decreased in 256, 512 and 1 024 mg•L⁻¹ BAEB group. The intracellular glycerol content of C. albicans was significantly increased in 512 and 1 024 mg•L⁻¹ BAEB group(P<0.05). The gene HOG1 associated with intracellular osmotic pressure of C. albicans was down-regulated by 9.1, 9.3 and 5.5 times, respectively. C. albicans with red fluorescent were increased significantly in 512 and 1 024 mg•L⁻¹ BAEB group. The peak area of ergosterol in the 1 024 mg•L⁻¹ BAEB group was 35.884 95, with a significant difference(P<0.05); ERG1, ERG2, ERG3, ERG4, ERG5, ERG6, ERG10, ERG11, ERG13, ERG24, ERG25, ERG251, ERG26 and UPC2 were down-regulated by 6.58, 4.89, 4.15, 9.24,3.41, 9.84, 3.08, 7.50, 5.53, 5.90, 2.45, 3.25,1.98 and 10.07 times respectively in 1 024 mg•L⁻¹ BAEB group. The study indicated that BAEB could inhibit ergosterol and its biosynthesis related genes expression in the cell membrane and inhibit the activity of C. albicans.

Effect of sodium houttuyfonate on symptom pattern of lung-Qi deficiency in rats induced by bacterialbiofilm infection.

OBJECTIVE: To investigate the in vivo inhibitory effects of sodium houttuyfonate (SH) on symptom pattern of Qi-deficiency in rats induced by infection of bacterial biofilm on rat respiratory tract. METHODS: Symptom pattern is a term used in Traditional Chinese Medicine (TCM) to define a cluster of symptoms in a medical condition. Based on the pattern, TCM therapies are administered. The symptom pattern used in this study was lung-Qi deficiency pattern identified in rats, which was induced by nasal intubation drip of Pseudomonas aeruginosa (P. aeruginosa) (two strains) to form bacterial biofilm on airway combined with stimulation of cold and fatigue. We measured the variations of the symptoms of the pattern, weight, spleen and thymus index, blood gas, lung bronchial tissue pathology and cytokine of rat in different treatments and control groups. RESULTS: The rats of SH-treatment groups had not showed typical symptoms comparing with model group in the early stage of infection. The weight, spleen and thymus index of the SH-treatment groups were significantly higher comparing with untreated model group. The SH-treatment groups also showed higher O(2) partial pressure and lower CO(2) partial pressure than model group. Furthermore, we found that the bronchopulmonary section of SH-treatment groups not showed typical pathogenic variation in model group. The comparison of cytokine concentration in different groups indicated that SH could prevent the over-production of cytokine to reduce the inflammation occurrence. CONCLUSION: In the early stage of airway infection by biofilm of P. aeruginosa, application of SH can prevent the occurrence of lung-Qi deficiency pattern.

[Sodium houttuyfonate inhibits virulence related motility of Pseudomonas aeruginosa].

Sodium houttuyfonate (SH) is a derivative of effective component of a Chinese material medica, Houttuynia cordata, which is applied in anti-infection of microorganism. But, the antimicrobial mechanisms of SH still remain unclear. Here, we firstly discovered that SH effectively inhibits the three types of virulence related motility of.Pseudomonas aeruginosa, i.e., swimming, twitching and swarming. The plate assay results showed that the inhibitory action of SH against swimming and twitching in 24 h and swarming in 48 h is dose-dependent; and bacteria nearly lost all of the motile activities under the concentration of 1 x minimum inhibitory concentration (MIC) (512 mg x L(-1) same as azithromycin positive group (1 x MIC, 16 mg x L(-1)). Furthermore, we found that the expression of structural gene flgB and pilG is down-regulated by SH, which implies that inhibitory mechanism of SH against motility of P. aeruginosa may be due to the inhibition of flagella and pili bioformation of P. aeruginosa by SR Therefore, our presented results firstly demonstrate that SH effectively inhibits the motility activities of P. aeruginosa, and suggest that SH could be a promising antipseudomonas agents in clinic.

Sodium houttuyfonate affects production of N-acyl homoserine lactone and quorum sensing-regulated genes expression in Pseudomonas aeruginosa.

Quorum sensing (QS) is a means of cell-to-cell communication that uses diffusible signaling molecules that are sensed by the population to determine population density, thus allowing co-ordinate gene regulation in response to population density. In Pseudomonas aeruginosa, production of the QS signaling molecule, N-acyl homoserine lactone (AHL), co-ordinates expression of key factors of pathogenesis, including biofilm formation and toxin secretion. It is predicted that the inhibition of AHL sensing would provide an effective clinical treatment to reduce the expression of virulence factors and increase the effectiveness of antimicrobial agents. We previously demonstrated that sodium houttuyfonate (SH), commonly used in traditional Chinese medicine to treat infectious diseases, can effectively inhibit QS-regulated processes, including biofilm formation. Here, using a model system, we demonstrate that SH causes the dose-dependent inhibition of AHL production, through down-regulation of the AHL biosynthesis gene, lasI. Addition of SH also resulted in down-regulation of expression of the AHL sensor and transcriptional regulator, LasR, and inhibited the production of the QS-regulated virulence factors, pyocyanin and LasA. These results suggest that the antimicrobial activity of SH may be due to its ability to disrupt QS in P. aeruginosa.

Antimicrobial effect of sodium houttuyfonate on Staphylococcus epidermidis and Candida albicans biofilms.

OBJECTIVE: To study antimicrobial effect of Sodium houttuyfonate (SH) on Staphylococcus epidermidis (SE) and Candida albicans (CA). METHODS: The prepared strain broths (OD,o=0.05) containing SE and CA were firstly used to test the minimal inhibitory concentrations (MICs) of SH, azithromycin (AZM) and fluconazole (FLU) by micro-dilution method. Then the biofilms of SE and CA were matured in 96-well plates, and co-cultured with SH, AZM and FLU for 1, 2 and 3 days to assess the antibiofilm efficacies of the agents with different concentrations by crystal violet staining method. At last, the treated biofilms of SE and CA by 2x MIC agents were observed by scanning electronic microscope. RESULTS: The MICs of SE and CA were 256 and 1024 microg/ml, respectively. After the 1st, 2nd and 3rd day of medications, the suppressions of biofilm were about 60% (P < 0.01), 76% (P = 0.000) and 75% (P = 0.000) by 2 x MIC SH, the suppressions of biofilm were about 90% (P = 0.000), 88% (P = 0.000) and 90% (P = 0.000) by 2 x MIC SH, which could be testified by scanning electron microscope results. However, the inhibitions of biofilm attachment had no significant difference for SE by SH and azithromycin and CA by SH and fluconazole. CONCLUSION: SH had widely anti-pathogenic effect on pathogenic biofilm formation of either bacteria or fungus, had more influence on enclosed cells of SE and CA than the traditional antibiotics, revealing its target might be the extracellular polymeric substances, and was more active to inhibit the growth of CA than SE.