Experimental Study Of Inhibition Of Sodium Nitrite On Trichophyton Rubrum

Objective: Superficial fungal infections are the most common and frequently-occurring disease of the skin, in which Trichophyton rubrum is one of the most common dermatophytes and the main pathogen in superficial mycoses. Trichophyton trubrum belongs to relatives of dermatophytes, which is the main violations of human skin, nail (toenail),sometimes violates peoples'hair .It is reported 90% of the chronic dermatophytes infection is caused by Trichophyton rubrum. At resent Trichophyton rubrum not only cause skin infection, but also cause deep tissue infections, morbidity is also increased year by year.Fungal diseases which is caused by Trichophyton rubrum are more difficult to heal and easy to relapse and re-infection, resulting in a decline in quality of life of patients.Trichophyton rubrum infection in patients with superficial mycoses choose topical antifungal agents, relatively safe, cheap, convenient, but because of issues such as patient compliance, drug therapy alone is often a time-consuming, prone to drug resistance phenomenon.United agents can improve efficency and the cure rate, shorten treatment time. A large number of studies show that NO which is a cytotoxic effector molecules as activated by macrophages and macrophag mediated suppress and anti-virus, bacteria, fungi and parasites, is an important body of anti-infection defense system. A large number of researches have shown that fungal infections and immune status are closely related, and NO can directly affect the body's specific immune and non-specific immunity. Many researchers in vitro experiments and animal models have found that NO play an important role in the immune system. Tong panzhuo et al research 30 cases of tinea pedis and 35 cases of onychomycosis patients′serum levels of NO and iNOS, compared with the normal group in which the patient group was lower than the level of the control group, and the level of NO and iNOS showed a positive correlation in a straight line. Tinea pedis and onychomycosis patients with low serum levels of NO and iNOS confirmed that NO play a role in fungi immunity.Sodium nitrite is a chemical substance which is easy to get electron to form NO in the acid form. Group abroad in recent years on the bactericidal action of sodium nitrite in vitro studies carried out, when the pH was 4.4 , 5 ~ 10 mmol / L of nitrite compounds can inhibit more than 95% of the yeast growth, which are also observed under the same conditions that acidification of nitrite compounds can kill other fungi and bacteria. At the same time, foreign study also confirmed the acidic environment of nitrite on the yeast, dermatophytes have strong killing effect. Topical application of acidified nitrite in onychomycosis patients for 16 weeks which is caused by Trichophyton rubrum, more than 90% of patients converted to negative culture results; application 3% nitrite cream in the treatment of tinea pedis for 4 weeks, 19 patients complete course of treatment in which 18 were completely cured. These studies confirmed that acidification of nitrite complex is an effective anti-fungal agent, but have no report about sodium nitrite inhibition of Trichophyton rubrum and ultrastructure observation.MTT is an inhibitory rate of cells was determined approach to reflect the numerical form of drugs on inhibitory cells. In recent years, MTT was applied to Candida, Aspergillus and other fungal drug susceptibility testing, proved to be a quick and easy Antifungal Susceptibility precise method to determine the end of the experiment. In our study, Trichophyton rubrum culture for 96 hours which contains different concentrations of sodium nitrite-based medicine in Sabouraud liquid, then use MTT research by measuring the absorbance at 530nm to quantify the value of sodium nitrite and the inhibition of Trichophyton rubrum. At the same time, by scanning electron microscopy, transmission electron microscopy observation of different concentrations and different times of sodium nitrite on the ultrastructure of Trichophyton rubrum for clinical application of laboratory based on sodium nitrite.Materials and methods:1 Choice fungal strains: Strains of Hebei Medical University for the second hospital to preserve the typical fungi Trichophyton rubrum, isolated from tinea corporis,tinea corporis,tinea pedis,onychomycosis and other superficial mycoses patients. According to the characteristics under the microscope ,Sabouraudand potato medium colony characteristics of identification, selection of Trichophyton rubrum 30.2 MTT method for different concentrations of sodium nitrite inhibition of Trichophyton rubrum: the application of different concentrations of MTT determination of sodium nitrite at 530nm to quantify the absorbance values of the inhibition of Trichophyton rubrum.3 sodium nitrite on the ultrastructure of Trichophyton rubrum impact:3.1 Scanning electron microscopy: To observe the different concentrations of sodium nitrite (160mmol / L, 80mmol / L, 40mmol / L, 20mmol / L) effect 96 hours to ultrastructure of Trichophyton rubrum and 160mmol / L sodium nitrite at different time (48h,72h,and96h) to ultrastructure of Trichophyton rubrum. At the same time they are equipped with the normal control group.3.2 Transmission electron microscopy: To observe the 80mmol / L sodium nitrite role of 96 hours to ultrastructure of Trichophyton rubrum the impact of drug-based growth does not add the control group.4 Statistical Methods: We use SPSS13.0 statistical software for data processing,one-way ANOVA comparison of the inhibition rate of different concentrations, and further analysis of the use of LSD test, P <0.05 for the difference was significant. Results:1 MTT results which sodium nitrite effect Trichophyton rubrum 96h:The concentration of sodium nitrite: 160mmol / L,80mmol / L,40mmol / L,20mmol / L,10mmol / L,5mmol / L,2.5mmol / L,1.25mmol / L, the role of 96 hours of Trichophyton rubrum to inhibition rates were as follows: (73.62+9.7%),(71.93+1.47%),(73.53+1.14%),(70.80+1.50%),(68.67+1.27%),(41.76+1.53%),(29.59+1.90%),(16.98+1.82%). With sodium nitrite concentration decreased, Trichophyton rubrum inhibition rate gradually reduced; by one-way ANOVA P <0.05, the difference has statistical significance. The results of this comparison further: 160mmol / L, 80mmol / L, 40mmol / L, 20mmol/L, 10mmol / L sodium nitrite to inhibit the rate of Trichophyton rubrum by comparing P>0.05,no significant difference; the rest of comparison between the concentration of both P <0.05, there was a significant difference.2 Scanning electron microscopy results:2.1 The role of different concentrations of sodium nitrite 96h ultrastructure of Trichophyton rubrum impact:No nitrite in normal control: good mycelial growth, surface smooth plump, uniform thickness; 20 mmol / L and 40 mmol / L sodium nitrite role of 96 hours: no significant changes in mycelial morphology normal, smooth surface; 80 mmol / L sodium nitrite role of 96 hours: mycelial surface in small depressions, mycelium thickness varies, the surface is not smooth; 160 mmol / L sodium nitrite role of 96 hours: mycelial deformation and expansion, varying thickness, a narrow dry mycelium department appears to undermine and fracture; at 80 mmol/L ~ 160 mmol/L range of concentrations of sodium nitrite as the increase in the mycelial form of the impact of the more obvious changes.2.2 160 mmol/ L sodium nitrite different time ultrastructure of Trichophyton rubrum impact:160 mmol /L sodium nitrite for 48 hours: mycelium did not change significantly, thickness is even, the surface is not smooth; 160 mmol/L sodium nitrite for 72 hours: mycelium thickness uneven surface less smooth, appeared to narrow the fault cleft department; 160 mmol /L sodium nitrite role of 96 hours: mycelium deformation and expansion ,varying thickness, a narrow dry mycelium department appears to undermine and fracture; sodium nitrite as the role of time, the impact on mycelial morphology gradually apparent.3 Electron microscopy results:No nitrite in normal control: a more complete picture of mycelium cut, thickness and uniformity, the nucleus can be seen in cytoplasm, mitochondria and vacuole structure; 80 mmol / L sodium nitrite role of 96 hours: cell swelling deformation, can be seen in the cell vacuoles structures, intracellular formation of high electron density of lectin block.Conclusion:1 Sodium nitrite can inhibit Trichophyton rubrum, when sodium nitrite concentration <10 mmol/L, with the concentration and time increase, the inhibition rate is gradually increase;when sodium nitrit concentration≥10 mmol / L,the inhibition rate does not change significantly.2 Low concentration (<40mmol/L)and short-term (72h) effect of sodium nitrite has no effect to the ultrastructure of Trichophyton rubrum .Long time high concentration and the role of sodium nitrite can affect the ultrastructure of Trichophyton rubrum, showing hyphal morphological changes and cell structural changes, with the role of concentration and time increase, the gradual changes in hyphal ultrastructure significant.