| The research purposeTo explore the Virological and Immunologic mechinism of TCM herbs treating the SARS. The virus model employed the Fowl Infectious Bronchitis, and the ARDS model was used to explore the immunologic mechanism of SARS. 1, To inspect the methods of the IBV and detectation of that virus, as wellas observing the antiviral function in vivo. 2, In vitro observe the antiviral and viral inhibition function of JiaweiShengjiangsan, and by employing the virus plaque bacteriophage toquantitatively estimate the antiviral function of that formula. 3, To build up the ARDS model of the mice and to estimate the amendment functionof the Jiawei Shengjiangsan on the indexes of blood gas test. 4, To approach the regulating function the Jiawei Shengjiangsan on the oxygenfree radical and inflammatory medium of mice ARDS modle and reveal it smechinism. 5, To discuss the influence of the Jiawei Shengjiangsan on the T lymphocytesubgroup of the mice ARDS model and reveal the immune regulating functionof it.The step of experiments 1, The passage of IBV and the detection.2, The influence of Jiawei Shengjiangsan on the IBV model in vivo. 3, The virus plaque bacteriophage test of the IBV.4, The gas and blood test, lung index and parquat LD50 of normal mice.5, The build up of the mice ARDS, the acute toxicity testing of the JiaweiShengjiangsan as well as the influence of the Jiawei Shengjiangsan on the mice ARDS blood and gas.6> The influence of Jiawei Shengjiangsan on MDA, SOD, NO. 7^ The influence of Jiawei Shengjiangsan on IL—1 P , IL-2, 1L-6, IL-8 and TNF-a. 8^ The influence of Jiawei Shengjiangsan on the mice T lymphocyte subgroup, methodK IBV innoculation , passage/the interferring of NDV as well as dwarf embryo test were all based on the method of Animal Virology. RT-PCR test was . performed under the instruction of the KIT. End product was examined by agarse gel electrophoresis, and the gel was test by ultraviolet, and the result was recorded by mini-visionaryTmgel imaging system. 2, IBV allantoic fluid, after freeze thawing driping into the binoculus and birhinia of chicken. And the chicken were divided into normal, model, positive control, herb big dose, herb middle dose and herb small dose groups radomly. 10 hours after the drug applying, The drugs was concentrated as the following ratio:the big dose group was 2:1, middle dose group was 1:1, small dose group was 1:2. Each mouse was given 0. 3ml for each adiministration and twice a day. Positive control group was given ganciclovir lOmg/kg fiat ope aquae destillatae, twice administration per day, each for 0.3ml. And for normal group and model group were given equal volume distilled water, treat the chicken by drugs. The experiment lasted for 9 days, the indexes including the symptoms, urination, defecation and death ratio.3> Chick embryo fibroblast(CEF) was cultured. 24 hours later, cells adherent to the plate, formed a compact monolayer cells. Chicken lung and airtube was grinded and diluted by saline water, centrifuged the mixtur, added antibiotics in the tube(final concentration 104U/ML), and dilute the mixture by 10"'~10"9 dilute strength. Added the mixture to the CEF, and move the cells into plate, high glucosenutrient medium, 1% fetal bovine serum, 5%C02 37*C incubator. After 48 hours, the cells are growing into a layer, use the haustorial tube to change the medium to 10ml medium without serum, standing in 37'C for lh. Put the plate into 5%C02 37°C incubator. After 24 hours count the- plaque.4^ Fix the mouse in the left hand, prick the injector which was treated by previously into the heart of the mouse. Draw off 500ul arterial blood, and sent to gas and blood test directly. Cut the chest of the mice and weighthe lung. The lung index was counted by the lung weight/body weight X 100. LD50 of parquat was based of the methods of Professor Xie Yangming, to divide the mice into 5 groups as following:48. 64 mg/kg, 61.44mg/kg, 76. 8mg/kg> 96mg/kg> 120mg/kg^ 150mg/kg. Administration once, obeserve for 14 days, count the death mice everyday, and the mice dead in 24 hours were exceptions. Using the Karber' s method to analysis the data.5, (l)The construction of the ARDS model: animals are grouped into 5 groups, they are normal group, 1 day after drug administration, 2 days after, 3 days after and 4 days after. Parquat was administrated 0. 18ml once for each group. And blood gas was tested everyday and analysis everyday. (2)The acute toxicity testing of Jiawei Shengjiangsan: animals are grouped into 4 groups, they are blank control group, big dose, middle dose and small dose group. Mice was fasting diet (applying water) 12h before the drug administration. 0. 3ml herbs was givent to the mice by oral administration once. And the blank control group was given distilled water. Oberserve for 1 week, twice a day on the morning and afternoon both. Record the activity and death of the mice, and the autopsy was performed on dead mice promptly. (3) The influence of Shengjiangsan on the gas and blood: animals were divided into blank control group, model group, and herb big, middle, small dose group. With the exception of normal control group, other groups were given parquate 0. 18ml once. 10 hours later treated these mice with herbs.3 days later, testing arteries blood gas. After the mice were killed, the lung was cut into 5mm size pieces and put it into 10% formaldehyde solution, routine anhydration, paraffin imbedding, HE dyeing, slicing and observing.6> Animals were divided into 5 groups, they were normal group, herb big, middle, and small group. The animal model and treatment were built and treated as previously introduced.SOD, MDA, NO were tested based on the introduction of the kits. Calculate by the formula.7, Animal group, model building and the drug administration was the same as the previous. IL-1 3, IL-2, IL-6, IL-8 and TNF-ct was tested follow the kit instruction. The data were calculated by the formula.8n Animal group, model building and the drug administration was the same as the previous. After the sacrificing of the rats, the blood was collected by the EDTA treated tube, the blood was treated with anticoagulation agents. 50 u lrat blood sample mixed with CDVT, CDVT, CDg'T monoclonal antibody each2. 5ii lwhich were bought from Jinmei Biotechinich company. After the samples were incubated in room teperature for 30 minutes, add haemolysin, 10 minutes later, centrifuge the samples at the speed of 1000G for 5-10 minutes, and wash the sample with PBS, centrifuge the sample for 5 minutes. Test the sample on the flow cytometry. Result1% IBV was blind passaged for 3 generations, NDV co-intervention determine the titre:the virulence of IBV is between 10"5-—10"6. The potency of NDV is 1:1024. Dwarf embryo test showed the EID50 was 0.2ml 10'566.This result conform with the NDV co-intervention test. Detect IBV by RT-PCR, successfully obtained 1404bp target gene. It is the same as expected. The results confirmed the virus was passaged was IBV.2^ The chicken are the normal on the firstday of the virus attacking.Since the second day, the symptoms like languor and standing still appeared. Since the third day, syptoms became obvious like languor, cough, scratch the nose, difficult breath, sneez .standing still, loose plumery, foodtakin reduced and with tracheal rale. Some even showed anhelation. 3-7days after the virus attacking was the syptom manifest period, since 8 days after, the symptoms decreased, and the chicken became active. The first dead chicken was observed on the 3rd day after. And 3-5 days after was the death peak, most of the dead chicken appeared during these period of time. Since the 6th day after, the death rate reduced and no death on the 8th day after. 3^he fibroblast culture adopted the halp-embryo method. Culture successfully. 24 hours culture before the next experiment. Cell growth in prosperity arid in good shape. After the virus was planted, the plaque was formed. The virus was diluted in 9 degree of concentration 1O''~1O"9. In 10'l~10"6 concetration, there is significance between the model group and the treat group. When the dilution degree went to 10"7, the plaques became too rare to be counted. So the result of the 10"7— 10"9 were ignored.4, Normal NIH mice, the arterial blood PaO2 (KPa) is 10. 77—14.83(12. 07±l. 27), PaC02(KPa) is 3. 05-6. 26(4. 58 + 1. 02), the lung indexe(%) is 0. 41-0. 76(0. 58 ±0.099), oral PQ LD50 is 87.096mg/kg.5> 2 days after the virus attaching, thePaO2, PaC^/FiOj began reducing obviously, PaC02 raise significantly, compared with normal group, (P<0. 01). In previous test showed that the indexes were peak at the 3rd day, so chose 3 days asthe total experimental time. After the treatment of herbs,the PaO2, Pa02/Fi02 were raised, and PaC02reduced effectively. Big dose group showed better result than middle,also the middle group was better than the small group. The lung histological test showed it was an acute interstitial pneumonia. After the treatment the hyperaemia manifestly ameliorate, phlegmasia cells infiltrating mitigate, and alveolar effusion reduced. The acute toxicity testing of Jiawei Shengjiangsan showed 1 week after the drug applied, the animal showed no difference with the normal mice, no death case, no weight reducement and the raio of the body growth is almost the same. 6n SODrthe model group showed a sharp reducement in this index, compared with normal group, (P<0.01). After the treatment, this index raised and compared with model group,the result was significant;MDA: the model group showed a sharp increase in this index, compared with normal group. After the treatment, this index droped and compared with model group, the result was significant, (P<0. 01);NO: the model group compared with normal group, P<0. 01. After the treatment, this index reduced and compared with model group, the result was significant (P<0.01).7^ IL—1 {3 : the model group is higher than the normal group (P<0. 01). The big dose and the middle dose group compared with the normal control group had no difference;IL-2: the model group was much lower than that of the normal group, P<0. 01, the middle and big dose group compared with the normal control group has no significance. Compared with the model group, showed significant difference (P<0. 01), and the small dose group compared with the model group, there was no significance;IL-6:the herb groups and the normal group compared with the normal group all the indexes are raised,P<0. 01, and all the three doses had significant difference with the model group, P<0.01;IL-8: the indexes were higher than the normal group significantl. After the treatment all the indexes reduced significantly compared with the model group;TNF-a: model group was higher than the normal group (P<0. 01).Each herb groups compared with the model group showed significant difference ( P<0.01). there was no differences between big, middle and small dose (P>0.05).8^ CD^ CDV CD8 reduced in the model, compared with the normal group, the difference is significant (P<0. 01 or P<0. 05). After the treatment the CD3, CD< raised compared with the normal group, there was no significance andcompared with the model group there is significant diference(P<0.01 or P<0. 05). After the treatment, CDs raised, compared with the normal group there was no significance but compared with the model group, big dose and middle dose has significantce(P<0. 05), and the small dose group had no significance. Showed that maybe the small dose group was not effective enough to raise the CD8. ConclusionK We have adopted the NDV co-intervention method, dwarf embryo experiment and RT-PCR to detect the virus which was blind passed for 3 generations. We have identified the virus was IBV and the EID50 of it was 10"566. 2> The Jiawei Shengjiangsan had the function to inhibit virus, increase the immunity of the chicken, improve the symptoms and reduce the death ratio as well as protect the animals from death. 3^ The Jiawei Shengjiangsan can reduce the virus plaque forming, suppress thevirus generating and antiviral function.4> The herbs detected was safe in the designing experimental dose rang. TheJiawei Shengjiangsan can ameliorate the mice model blood gas, raise theblood Pa02 and reduce PaC02 and can improve the mice ARDS model symptomsmand improve their pathological lesion.5^ The Jiawei Shengjiangsan had the antioxidation function and improve theirpathological lesion.6^ The Jiawei Shengjiangsan can increase the phagocytose function of the macrophage. And inhibite the macrophage secrete IL-l,TNF-a, and also reduce the IL-6> IL-8 quantity. Reduce the damage of the inflammation mediators as well as pneumochysis. Increase the secretion of the IL-2. This formula had an obvious function to regulate the immun system.7> The Jiawei Shengjiangsan can do retification of the disorder situation of T lymphocytes subgroup. And can enhance the T cells proliferation, the mice spleen secrete IL-2, Increase the cell immun function.
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