Study On The Antibacterial Effect And Mechanism Of Selenium Glycyrrhiza Uralensis Fisch Polysaccharide And Its Combined Antibacterial Drugs Against Gram-Positive Bacteria

Gram-positive bacteria are mostly septic bacteria,and antibiotics are commonly used for clinical treatment,leading to the frequent occurrence of bacterial drug resistance,and the research of new antibacterial drug preparations to slow down and eliminate the phenomenon of bacterial drug resistance has attracted the attention of scholars at home and abroad.Chinese herbal medicine occupies an important position in the history of medicine in China,with the advantages of green,low toxicity and non-pollution.As early as thousands of years ago,Chinese herbal medicine was used to treat bacterial diseases,so the research on the antibacterial effect of Chinese herbal medicine has become a craze.Objective:Study on the antibacterial effects of selenium Glycyrrhiza uralensis Fisch polysaccharide（Se GUP）,Glycyrrhiza uralensis Fisch polysaccharide（GUP）,and their combination with antibiotics on common clinical Gram positive bacteria,and to provide theoretical basis for further research on green non-polluting antibacterial agents and immune enhancers.Methods:（1）In vitro antibacterial activity study:Gram-positive bacteria sensitive to Se GUP were screened by the filter paper sheet method;antibacterial drugs with good effect on sensitive bacteria were screened by the drug sensitivity sheet;the minimum inhibitory concentration（MIC）of Se GUP,GUP and antibacterial drugs on sensitive bacteria were measured by the minimum twofold dilution method;the minimum bactericidal concentration（MBC）of Se GUP,GUP and antibacterial drugs were measured by the checkerboard method;the fractional inhibitory concentration index（FICI）between Se GUP,GUP and antibacterial drugs was measured by the checkerboard method.and their combined antibacterial drugs were mixed with sensitive bacteria and the bacterial growth rate was plotted.（2）Study of in vitro antibacterial mechanism:The screened sensitive Gram-positive bacteria were mixed with Se GUP,GUP and their combined antibacterial drugs,and the contents of AKP,β-gal and protein in the culture medium were determined by ELISA,the intracellular ATPase activity,ATP content,TOPⅠand Mun of the bacteria were determined,and the intracellular DNA content was detected by bacterial DNA extraction kit.Then the effect of antibacterial drugs on the biofilm formation of Gram-positive bacteria was observed by crystalline violet staining method.（3）In vivo antibacterial activity study:The minimum lethal dose（MLD100）was determined to kill all the infected mice;the mice were given antibacterial drugs before and after the attack,and the lesions were made into paraffin sections and the pathological changes were observed under microscope to investigate the prevention and treatment effects of Se GUP,GUP and their combined antibacterial drugs on the infected mice.（4）Immunomodulatory effects on immunosuppressed mice:Se GUP and GUP were given to immunosuppressed mice,their daily weight and status changes were recorded,the immune organ index was measured,the content of IL-2,IL-10,IFN-γand TNF-αin the serum of mice was determined by ELISA,the changes in blood cell content of mice were analyzed by automatic hematocrit analyzer,and the changes in blood cells were calculated by hematocrit.Counting method was used to calculate the number of nucleated cells in bone marrow of mouse femur,and the absorbance value at OD_260nm.Results:（1）In vitro antibacterial activity study:Listeria monocytogenes and Streptococcus pneumoniae were highly sensitive to 480 mg/m L Se GUP,and Streptococcus pneumoniae was moderately sensitive;Streptococcus pneumoniae,Listeria monocytogenes and Streptococcus pneumoniae were moderately sensitive to 480 mg/m L GUP;the effect of Se GUP was better than that of GUP as shown by MIC,MBC and FICI.Se GUP,GUP and their combined antibacterial drugs inhibited the growth rate of bacteria,and the inhibitory effect of Se GUP was stronger than that of GUP at equivalent concentrations,and the effect of combining with antibacterial drugs was also better than that of GUP.（2）Research on in vitro antibacterial mechanisms:Se GUP,GUP on AKPβ-The leakage of gal and protein has a promoting effect,which indicates that Se GUP and GUP can destroy the integrity of bacterial cell wall and membrane,and can also reduce the content of DNA,ATPase,ATP,TOPⅠ,Mun in cells.Compared with the blank control group,the contents of Se GUP,GUP120 mg/m L or 60 mg/m L groups and their combined antibacterial groups are significantly reduced（P<0.05）.（3）Research on in vivo antibacterial activity:Different concentrations of Se GUP and GUP administered to mice before and after the attack can significantly reduce the degree of inflammation and improve the changes in pathological tissue structure in mice.The combined use of antibacterial drugs has a better recovery effect on the tissue structure of mice than the use of various drugs alone.（4）（4）Research on the Immunomodulatory Effect of Se GUP and GUP on Immunosuppressive Mice:Se GUP and GUP can gradually normalize the body weight of immunosuppressive mice,and IL-2 and IFN-γ、TNF-α,The content of IL-10 significantly increased（P<0.05）,leading to a gradual regression of IL-10 content to the blank group.The Se GUP 300 mg/kg and150 mg/kg groups showed a significant increase in lymphocytes,white blood cells,and red blood cells（P<0.05）,significantly restoring the content of mouse bone marrow nucleated cells and DNA.Conclusion:Se GUP and GUP have good antibacterial activity against Streptococcus pneumoniae,Listeria monocytogenes and Streptococcus pneumoniae in vivo and in vitro,and can improve the changes of inflammation in the lung,spleen and breast of mice.Se GUP and GUP can not only enhance the antibacterial activity but also reduce the dose of antibacterial drugs when combined with kanamycin hydrochloride,doxycycline hydrochloride,ceftriaxone sodium and flupenthixol.The high,medium and low doses of Se GUP could signi FICI antly improve the immunity of the organism,with the best effect at 300mg/kg,which may be one of the reasons for its ability to enhance the antibacterial activity of antibacterial drugs.The antibacterial effect of Se GUP in vivo and in vitro at the same dose was stronger than that of the GUP group,and the effect of Se GUP combined with antimicrobials was better than that of GUP combined with antimicrobials.