Isolation Of Rat Peritoneal Mast Cell And Houttuynia Cordata Thunb Injection's Stimulation On The Cells To Degranμlate

1 Objective:Traditional Chinese medicine injection is based on Traditional Chinese medicine theory, using modern science technology, and distilling efficient substances from Traditional Chinese medicine in formulas, which are prepared to be injections. The study of Traditional Chinese medicine injection was not long before, and the technology hasn't been perfect yet, so along with widely using Traditional Chinese medicine injection to treat patients, the reports of untoward effect always was happened. National center for ADR monitoring, China analyzed cases and reported that anaphylactic reaction, even anaphylactic shock could happened during Houttuynia Cordata Thunb injection, and notified nationally to stop using using 7 Traditional Chinese medicine injections including Houttuynia Cordata Thunb injection. Houttuynia Cordata Thunb injection is colorless transparent injection that was prepared by distilled Houttuynia cordata. Pharmacology tudy of Houttuynia Cordata Thunb injection proves that it has effect of antibiosis and enhancing immunity. Number of reports about side effect of Houttuynia Cordata Thunb injection was 116 from Jan 1994 to Aug 2005, number of cases was 265. Anaphylactic reaction caused by Houttuynia Cordata Thunb injection could be appearance of respiration system and circulation system et al, and which is anaphylactic shock at worst and 40.3 % of which is appearance of respiration system. Analyzing cases of;anaphylactic reaction caused by Houttuynia Cordata Thunb injection,49% of patients had definite no allergy history, in only a few patients'serum the quantity of IgE increases. So these symptoms were not made by anaphylactic reaction but anaphylactoid reaction. Anaphylactoid reaction has similar appearance as anaphylactic reaction, but isn't immune reaction, which could happen without special antibody.Studies about anaphylactoid reaction caused by Houttuynia Cordata Thunb injection were most carried in vivo. It is in vivo that could provide real environment of injection's reaction, while, process of research in vivo is complex and easy to be affected by other factor. Research in vitro can make up for a deficiency of research in vivo, and is more sensitive, which is on level of cell. In this study, Houttuynia Cordata Thunb injection was directly action on mast cell to make a pilot study anaphylactoid reaction caused by Houttuynia Cordata Thunb injection.Mast cell is the major active cell in anaphylactoid reaction and anaphylactic reaction, which could degranulate to release lots of inflammatory factors and induce allergy. Mast cell is import carrier of anaphylactoid reaction. However mast cell strain in ATCC(American type culture collection)are all mouse's cell strain. Studies about rat's mast always has RBL-2H3 cell instead, which cell strain was basophil fibroblastoma cell that is not suitable for mast cell study. Many studies could be brought about by isolation mast cell from rat. Lennart invented a method of isolation rat peritoneal mast cell(RPMC) by density gradient centrifugation. In this study, optimized method on the base of Lennart's method was used to isolate RPMC.2 Methods2.1 Isolation of RPMCAfter ether anaethestizing SD rat, cut its carotid arteries to let it bleed till death, then injected 12ml Tyrode solution Ca2+ free into its peritoneal cavity. After gentle massage for 3min, peritoneal wash solution was recoveried, which was centrifuged then. Supernatant was discarded, and 0.75ml Tyrode solution was used to resuspend cells. Mixed cell suspension with 3.5ml percoll solution of 1.110g/ml, and centrifuged the mixture at 130g at 15min. Then the uppest 2.5ml solution, and the rest cell suspension contained mast cell in a purity, which should be washed 3 times. In this study, optimized method of isolation RPMC was brought forward, and staining toluidine blue indicator solution and trypan blue indicator solution indicated that optimized method is better than the old one. Cell viability was more than 99%, Cell purity was more than 90%. Electron micrograph of the cells shows that isolated cells were mast cells. C48/80 stimulated isolated cells to check the activity of degranulation as mast cell of these cells.2.2 Houttuynia Cordata Thunb injection's action on RPMC directly.RPMCs stimulated directly by four times diluted Houttuynia Cordata Thunb injection, RPMCs acted by Trition X-100, RPMCs stimulated by C48/80 and RPMCs not stimulated were put in incubator of 37℃,5%CO2 for 30min. Then the rate of release of hexosaminidase was measured.2.3 The stimulation on mast cell of injections of different concentrationHouttuynia Cordata Thunb injection was twice, four times, six times and eight times diluted into different concentration. These different injections and trition X-100, C48/80 acted on RPMCs, then these RPMCs and RPMCs not stimulated were put in 37℃,5%CO2 for 45min.45min later, measure the rate of release of hexosaminidase of the groups.Houttuynia Cordata Thunb injection was twice, four times and eight times diluted into different concentration. These different injections and C48/80 acted on RPMCs, then these RPMCs and RPMCs not stimulated were put in 37℃,5%CO2 for 45min.45min later, measure quantity of released histamine by RPMCs in different groups.2.4 The action on mast cell of Houttuynia Cordata Thunb injections of different lot numberHouttuynia Cordata Thunb injections of different LOT number, which were all diluted 4 times, and trition X-100, C48/80 acted on RPMCs, then these RPMCs and RPMCs not stimulated were put in 37℃,5%CO2 for 45min.45min later, measure the rate of release of hexosaminidase of the groups.Houttuynia Cordata Thunb injection s of different LOT number, which were all diluted 4 times, and C48/80 acted on RPMCs, then these RPMCs and RPMCs not stimulated were put in 37℃,5%CO2 for 45min.45min later, measure quantity of released histamine by RPMCs in different groups.3 Resμlts3.1 Isolation of RPMCOptimized method of isolation RPMC was brough forward, and staining toluiding blue indicator solution and trypan blue indicator solution indicated that optimized mehode is better than the old one.Cell viability was more than 99%, Cell purity was more than 90%. Electron micrograph of the cells shows mast cells' appearance. RPMCs stimulated by C48/80 released much more hexosaminidase,the rate of which was 50.91%±3.13%, and more histamine that was 2.274±0.04lng/ml than the cells not stimulated released,2.59%±0.10% and 0.013±0.013ng/ml,repectively.3.2 Houttuynia Cordata Thunb injection's action on RPMC directlyIn positive group the rate of release of hexosaminidase was 51.49%±1.91%, while in negative group was 3.01%±0.3%, and in injection group,in which group, cells were stimμlated by injection,the the rate of release of Hexosaminidase was 8.20 %±0.88%, having significant differce from the rest groups.3.3 The action on mast cell of injections of different concentrationRPMCs stimulated by Houttuynia Cordata Thunb injections diluted twice released 35.83%±5.84% hexosaminidase, while injections diluted eight times released 12.19%±1.11% hexosaminidase. RPMCs stimulated by Houttuynia Cordata Thunb injections diluted twice released 4.26±0.51ng/ml histamine; injections diluted eight times released 2.58±0.02ng/ml. There were significane differences between groups. The degranμlation was related to the concentration of the injection, the higher concentration of injection group has a higher rate of release of hexosaminidase and histamine than the lower one.3.4 The action on mast cell of Houttuynia Cordata Thunb injections of different lot numberRPMCs stimulated by Houttuynia Cordata Thunb injections of 100701 LOT number released 19.88%±4.36% hexosaminidase and 2.26±0.37ng/ml histamine; RPMCs stimulated by Houttuynia Cordata Thunb injections of 100805 LOT number released 16.25%±4.03% hexosaminidase and 2.11±0.28ng/ml histamine. There was significant difference in the rate of release hexosamidase by RPMCs between some groups of injection of different Lot number, not in the rest.There was no significant difference in quantity of histamine released by RPMCs between the groups of injection of different Lot number.4 Conclusions4.1 Optimized method of isolation RPMC is feasibleUsing optimized method of isolation RPMC coμld isolate cells of good viability and high purity of mast cell, which coμld be used in viarious research about mast cell.4.2 Houttuynia Cordata Thunb injection can cause anaphylactoid reactionHouttuynia Cordata Thunb injection acted on RPMCs and made RPMCs degranμlate to release more hexosaminidase than RPMC added nothing (blank control), which indicated that Houttuynia Cordata Thunb injection can cause anaphylactoid reaction.4.3 The action on mast cell of injections of different concentration is differentHouttuynia Cordata Thunb injection of High concentration caused RPMCs released more hexosaminidase and histamine than injection of low concentration.4.4 The action on mast cell of injections of different LOT nμmber is similarHouttuynia Cordata Thunb injections of different LOT nμmber, which were gifts from GUANGDONG BOLUO PIONEER MEDICINAL GROUP CO.,LTD caused RPMCs released similar hexosaminidase and histamine.The research on anaphylactoid reaction caused by Houttuynia Cordata Thunb injection in vitro, costs less time and is much easier than research in vivo. In this study, beside introducing a optimized method of isolation RPMC, anaphylactoid reaction caused by outtuynia Cordata Thunb injection was discussed in different ways, including the quantity and the different LOT nμmber's affection. This study offer reference to apprise anaphylactic and anaphylactoid reaction caused by Traditional Chinese Medcine, which will help in clinical using Traditional Chinese Medcine including Houttuynia Cordata Thunb injection safely.