Screening Of Chinese Medical Formula For Curing Streptococcus Suis Meningitis And Initial Study Of Its Action Mechanism

Swine streptococcosis caused large serious lost in the worldwide. The antibiotic resistancenot only induced the weakened efficacy to this disease, but also caused the environmentalpollution and food safety issues. Therefore, the more secure and effective measure to prevent thisdisease is needed.. Traditional Chinese medicine has been used to control animal diseases withextensive experience in China for a long history. The purpose of this study is to screen effectiveChinese medical formula to swine strepococcosis with differentiation of syndromes under theguidance of the theory of traditional Chinese veterinary medicine, further to elucidate itsmechanism of action from the cellular and molecular level, laying the foundation for its clinicalapplication.S. suis serotype2(SS2) strain CVCC606is purchased from China Institute of VeterinaryDrug Control. It is identified by means of morphological observation and polymerase chainreaction (PCR), and the basic characteristics and virulence of this strain is also examined. Theresults showed that the ascending order of various conditions affecting the hemolytic activity ofstrain CVCC606is: the proportion of inoculation, culture time, pre-culture time and pH of theculture medium. The optimal generation conditions of suilysin (SLY) of S. suis serotype2(SS2)strain CVCC606is: pH7.3of the culture medium,3%of inoculation,18h of pre-culture time,and12h of culture time.To obtain the higher purity and activity of the SLY, SLY was purified using differentfiltration steps, fractional precipitation with ammonium sulfate, and Sephadex G-100columnchromatography. The purified SLY exhibited a specific activity of4701.46HU/mg, therecoveries of SLY is2.88%, and the fold of purification is761.99.Symptoms of meningitis was observed in Sprague–Dawley (SD) rats after infected with S.suis by intraperitoneal injection, the LD50(median lethal dose) is3.4×109CFU/mL of each rat.Rat model of S. suis meningitis is successfully established by intraperitoneal injection of0.5LD50of SS2strain CVCC606. The significant clinical S. suis meningitis signs is observed in all challenged rats, and necropsy and histopathology examination is consistent with thepathological changes of S. suis meningitis. Bacteria can be isolated from the liver, spleen andcerebrospinal fluid (CSF) of challenged rats.According to the theory of traditional Chinese veterinary medicine, heat syndrome isanalyzed by eight-principle syndrome differentiation, both nutrient-phase syndrome andblood-phase syndrome is analyzed by weiqiyingxue syndrome differentiation, heat intonutrient-phase and blood-phase, poison into pericardium, and liver wind syndrome is analyzedby Zangfu syndrome differentiation. The therapeutic principle is to clear heat, cool the blood,and calm liver wind. Four decoctions (Huanglian Jiedu decoction, Qingying decoction, XijiaoDihuang decoction and Gouteng Xijiao Dihuang decoction) were chosen for the treatment. Theresults showed that Gouteng Xijiao Dihuang has better efficacy. This decoction consists of100gof buffalo horn,150g of rehmannia,45g cortex moutan,60g of paeonia lactiflora, and60g ofuncaria rhynchophylla,1g of crude drug in1mL after decocting. Rats were treated with GoutengXijiao Dihuang decoction in intragastric administration in1mL of each, one time per day for5days.To study the mechanism of Gouteng Xijiao Dihuang decoction, we establish the model ofBrain Microvascular Endothelial Cells (BMVECs). RBMVECs is successfully cultured with themethod of twice digestion combined with twice density gradient centrifugation. Cells isconfirmed as endothelial cells by morphological observation and mmunofluorecent staining of F-Ⅷ Rag RBMVECs. Growth curve of RBMVECs was determined by MTT assay, indicating itspeak growth phase is from3d to7d.The results showed that there is no cytotoxicity when the concentration of Gouteng XijiaoDihuang decoction less than80μg/mL, the concentration of paeonol, catalpol and cholesterol lessthan100μg/mL, the concentration of paeoniflorin less than50μg/mL, and the concentration ofrhynchophylline and isorhynchophylline less than10μg/mL. And concentration of2μg/mLandabove SLY can cause damage and the abnormality of tight junction of RBMVECs, and thedamage to the cells is positive correlated with concentration of SLY and the prolonged durationof challenge.32μg/mL of SLY is chosed to establish the cell model of cell damage and tightjunction of RBMVECs for12h challenge. Cytotoxiciy induced with SLY and the abnormality of tight junction of RBMVECs can bereduced or eliminated with Gouteng Xijiao Dihuang decoction, paeonol, catalpol, paeoniflorinand cholesterol, rhynchophylline and isorhynchophylline. Protection of RBMVECs from damageand the increase permeability of the cells has great significance for the consolidation of BBB.IL-6can be produced by RBMVECs induced with SLY, and the quantity of IL-6productionis positive correlated with concentration of SLY and the prolonged duration of challenge.Gouteng Xijiao dihuang decoction and its active components could reduce the cytotoxicity ofsuilysin to RBMVECs, and suppress the IL-6release by RBMVECs induced with suilysin, aswell as correct NO and ET-1secretion imbalance, and prompting the ratio of NO and ET-1at ahigh state of equilibrium, which result in the the sensitivity threshold of the RBMVECs isenhanced, and the ability of resistance to injury of RBMVECs is improved.