Background C57BL/6(B6) is a kind of routine mouse specie used in experimental autoimmune uveitis (EAU) research.Previous studies showed that the pathogenesis of uveitis related to inflammatory cytokines secreted by different helper T(Th) cells.However,the interaction of different Th cells in EAU is unclear.Objective This study was to investigate the dynamic changes of inflammatory factors in the spleen and serum after immunization in EAU mice.Methods Forty-four SPF B6 mice were immunized by injection of interphotoreceptor retinoid-binding protein (IRBP) and complete Freund adjuvant (CFA) emulsion via caudal vein and footpad.Indirect ophthalmoscope was used to examine the eyes 3 times per week and the inflammatory response was scored based on Thurau's criteria.In the thirty day after injection,20 model eyes were extracted and the sections of eye tissue were prepared for histopathological examination.The spleens of model mice were enucleated before injection and 2,5,10,15,20,25,30 days after injection,and reverse transcriptase PCR (RT-PCR) was used to detect the contents of interleukin-17 (IL-17) mRNA,interferon-γ (IFN-γ) mRNA,tumor necrosis factor-α (TNF-α) mRNA and IL-10 mRNA,and the contents of IL-17,IFN-γ,TNF-α and IL-10 in model serum were assayed by ELISA in 24 model mice.The experimental protocol and use of the animals were approved by Ethic Committee for Care and Use of Laboratory Animals of Shandong University of Traditional Chinese Medicine.Results Mild inflammatory response was seen in 12 days under the indirect ophthalmoscope with the scores of 0.5.The inflammatory scores peaked in 13-15 days with the scores of 1.0 and alleviated after that with the inflammatory scores of 0.5 in 30 days after injection.The histopathological score was consistent with the clinical score in the models on the 30 days.The serum IL-17 content of model mice was (0.98±0.05) ng/L before injection and increased to (51.85 ±2.42) ng/L on the fifth day,and decreased to (4.01±0.06)ng/L on the fifteen day.But,the serum IL-17 level increased to (25.00±0.94)ng/L again on the 25th day,and then lowed to (6.01 ±0.21)ng/L 30 days after injection,showing a significant elevation in comparison with that of before injection (P=0.000).The serum IFN-γ content of the model mice was (1.02±0.09)ng/L before injection and increased to (50.54±0.48) ng/L on the fifth day,and (73.21±0.12) ng/L on the tenth day,and then it declined gradually until (5.15±0.18)ng/L in the 30th day,which was still higher than that of before injection (P=0.000).After injection of IRBP+CFA,the serum TNF-α level upregulated from the second day to fifth day with the peak values (134.25±0.59)ng/L,and declined to valley on 15th day.A repeat elevation of serum TNF-α level was found on the 20th day with the values (60.54±0.62)ng/L and followed by decrease till the 30th day,which was higher than that of before injection (P=0.660).Serum IL-10 was detectable in the tenth day and peaked on the 15th day.Then a slight decrease was seen till the 30th day,compared with before injection(P =0.000).The contents of IL-10 mRNA,IL-17 mRNA,TNF-α mRNA,IFN-γmRNA in mice spleens followed the same pattern with serum levels of their proteins.Conclusions IL-17,IFN-γ,TNF-α and IL-10 are key inflammatory factors of Th1,Th2 and Th17,they present with specific changes during EAU,it confirming that IFN-γ probably play a pathogenic role in EAU,IL-17 and TNF-α levels probably associated with the chronic and recurrent procedure of uveitis,IL-10 plays an inhibit role in EAU.

Background Lewis rat is a commonly used specie in experimental autoimmune uveitis(EAU).However,the characteristics of EAU,especially ocular uhrastructural change,are rarely reported.Objective This study was to investigate the inflammatory characteristics and ocular uhrastructure of EAU models in Lewis rat.Methods EAU models were induced in 12 SPF female Lewis rats(6-8 weeks old)by injection of complete Freund adjuvant(CFA) containing interphotoreceptor retinoid-binding protein (IRBP,1177-1191) and tuberculin (TB) into footpads,napes and back of the body,and 6 normal matched rats were used as normal controls.The diet and drinking,temperature and behavior acts of the rats were recorded during the observational duration.Ocular manifestations were examined under the slit lamp biomicroscope after modeling and scored.Eyeballs were obtained in 12 days after modeling for histopathological examination,and the ultrastructures of eyeballs were observed under the scanning electron microscope and transmission electron microscope.The use and care of the animals complied with Statement of ARVO.Results The food-intake was (190.00± 18.03)g in the model group,and that in the control group was (285.33 ±28.02) g,showing a significant difference between them (t =4.955,P =0.012).The drinking-water volume of rats was (241.67±t 18.56)ml in the model group,which was significantly less than (289.67± 18.18)ml in the control group,showing a significant difference between them (t =3.201,P =0.033).In addition,elevated temperature and tiredness were found in the model rats.Anterior chamber empyema,iris hyperemia and occlusion of the pupil appeared in the models on the 6th day and peaked on the 12nd day after immunized,with the inflammatory scores of 3.83±0.41.The infiltration of inflammatory cells were seen in anterior chamber,ciliary body and vitreous cavity under the optical microscope in the model rats.Scanning electron microscopy found uneven iris texture,coarse ciliary surface and loosen villi of retinal pigment epithelial (RPE) cells in the model rats.Under the transmission electron microscope,the infiltration of macrophagocytes on the iris,wrinkle sparse of ciliary epithelium,myeloid bodies in retinal Müller cells and vacuolus in mitochondria of RPE cells were exhibited.No obvious abnormality was found in the control rats.Conclusions Lewis rats are autoimmune status following injection of CFA with IRBP(1177-1191) and TB.The morphology and ultrastructure of eyeballs in the EAU rats can explain the finding of eyes.

A laccase gene (lacD) from the basidiomycete Trametes sp. 420 was heterologously expressed in Pichia pastoris in two ways, resulting in two recombinant enzymes of rLacDx with native N-terminus and rLacDe with eight additional amino acid residues at N-terminus. The yields of rLacDx and rLacDe in shaken-flask cultures after an 18-day growth were 1.21 x 10(5) u/L and 7.38 x 10(4) u/L, respectively, as determined with 2,2'-azinobis(3-ethylbenzothia-zoline- 6-sulfonic acid) (ABTS) as substrate. The yield of rLacDx was further increased to 2.39 x 10(5) u/L under high-density fermentation while the production process was decreased to 7.5 days. In addition, rLacDx and rLacDe exhibited similar enzymatic characters in oxidizing substrate guaiacol, and were stable at 50 degrees C and at a pH range from 3 to 10. However, the specific activity of rLacDx (1761 u/mg) for ABTS was higher than that of rLacDe (1122 u/mg), and the apparent Km value of rLacDx (427 microM) was less than that of rLacDe (604 microM).
Cloning, Molecular ; Fermentation ; Isoenzymes ; biosynthesis ; genetics ; Laccase ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Trametes ; enzymology ; genetics

ObjectiveTo study the effect of Xiaoyusan new formula on the articular cartilage of knee osteoarthritis (KOA) rabbits and its mechanism. MethodsA total of 42 New Zealand white rabbits aged 6 months were randomly divided into normal group, model group, ointment of Xiaoyusan group, and ointment of Xiaoyusan new formula group, with 10 rabbits in each group (the other 2 rabbits were used for model validation). Except for the normal group, the right knee joints of all rabbits in the other groups were prepared as KOA models according to the modified Hulth method. After 5 weeks of molding, the rabbits in ointment of Xiaoyusan group, ointment of Xiaoyusan New Formula group were given corresponding ointments for knee arthritis treatment, once a day, each time for 10 hours. After 2-week continuous administration and treatment, the knee joint cartilage of the four groups of rabbits was taken and the cartilage damage of each group was evaluated by Outerbridge grading method. The pathological changes of the cartilage, calcified layer and subchondral bone of the knee joint of rabbits in each group were observed by HE staining method under the light microscope, and the degree of cartilage degeneration was evaluated by Mankin's method. The expression of matrix metalloproteinase-13 (MMP-13) in the cartilage of rabbit knee joint in each group was deteced by immunohistochemistry. Results After the general observation of articular cartilage, the Outerbridge grading showed that the number of high-grade animals in ointment of Xiaoyusan group was reduced compared with the model group (P<0.05), and the number of high-grade animals in ointment of Xiaoyusan new formula group was also reduced (P<0.05) compared with ointment of Xiaoyusan group. HE staining showed that Mankin's scores of articular cartilage in the four groups ranked from high to low: model group (10.82±1.76), ointment of Xiaoyusan group (6.19±1.23), ointment of Xiaoyusan new formula group (2.64±1.18) and normal group (0.28±0.17). The difference among four groups was statistically significant (P<0.05). Immunohistochemical detection showed that the positive rates of MMP-13 expression in rabbit articular cartilage tissues in each group were (67.90±13.94)% of model group, (37.10±19.16)% of ointment of Xiaoyusan group, (13.60±3.10)% of ointment of Xiaoyusan new formula group and (3.20±2.39) % of normal group, ranking from high to low, and the difference among four groups was statistically significant (P<0.05). ConclusionXiaoyusan new formula can repair articular cartilage degeneration in KOA rabbits and decrease the expression of MMP-13 in cartilage, which may be one of the mechanisms of the treatment.

Objective:To discuss clinical effect of addition and subtraction therapy of Ditantang combined with Taohong Siwutang to cerebral infarction and syndrome of phlegm and blood stasis blocking collaterals during early recovery， and to study protection to brain nerve. Method:One hundred and fifty-two patients were randomly divided into control group （76 cases） and observation group （76 cases） by random number table， 71 patients in control group completed the therapy （5 patients were falling off， missing visit or eliminated）， and 70 patients in observation group completed the therapy. Both groups' patients got comprehensive rehabilitation measures. Patients in control group got Zhongfeng Huichun pills， 1.5 g/time， 3 times/day. Patients in observation group got addition and subtraction therapy of Ditantang combined with Taohong Siwutang in the morning and at night， 1 dose/day. The treatment was continued for 12 weeks. Before and after treatment， scores of degree of neurological deficit， Barthel （BI） index， Fugl-Meyer scale （FMA）， modified Rankin scale （MRS） and syndrome of phlegm and blood stasis blocking collaterals were graded. And levels of malondialdehyde （MDA）， glutathione peroxidase （GSH-Px）， superoxide dismutase （SOD）， advanced oxidation protein products （AOPP）， vascular endothelial growth factor （VEGF）， brain-derived neurotrophic factor （BDNF） and neuron specific enolase （NSE）. And cerebral hemodynamics were detected， and peak flow velocity （VS）， vascular resistance index （RI）， pulsatility index （PI） and cerebrovascular reserve function （CVR） were recorded. Safety was evaluated. Result:After the 6^th week and 12^th week of treatment， scores of degree of neurological deficit， BI， FMA， MRS， syndrome of phlegm and blood stasis blocking collaterals， AOPP， MDA， NSE， RI and PI were lower than those in control group （P<0.01）， levels of SOD， GSH-Px， BDNF， VEGF， Vs and CVR were higher than those in control group （P<0.01）. The clinical effect was better than which in control group （Z=2.109， P<0.05）. Besides， there was no adverse reaction caused by Ditantang combined with Taohong Siwutang. Conclusion:Ditantang combined with Taohong Siwutang can ameliarate the hemodynamics， reduce the lipid peroxidation damage， regulate the neurovascular repair factor， so it can promote the repair of nerve tissue and function， clinically reduce the degree of nerve function defect， improve the ability of daily life and exercise when it used to cerebral infarction and syndrome of phlegm and blood stasis blocking collaterals during early recovery， and it is good for clinical effect and safe using.