Effects Of Acupoint Injection With Self-blood On The Inflammation, Th2, Th1, Th17, And Treg's Cytokine,Transcription Factors, And Related Mrna In Asthmatic Rats

ObjectiveTo observe the effects of Acupoint injection with self-blood (AISB) on the asthmatic inflammation, Th2, Thl, Th17, and Treg's cytokine, transcription factors and related mRNA in asthmatic rats, we further explored the mechanism in which AISB works, comparing among other2clinical methods (normal saline point injection and Dexamethasone intraperitoneal injection) in a randomized, controlled study.MethodsAccording to the principles of Comparison, Randomization and blindness in statistics, we designed the one-factor-triple-level experiment proposal.48SD rats (SPF level, male,130～150g, supported by the lab animal center of Guangdong) were divided randomly into5groups:Normal group(Group A, no sensitization or treatment, n=8), Model group (Group B, Asthmatic model, n=10), NS group (Group C, Point injection with normal saline, n=10), AISB group (Group D, Acupoint injection with self-blood, n=10) and DXM group(Group E, intraperitoneal injection with Dexamethasone, n=10). We raised the rats and operated the experiment in a SPF level animal center (No.1Affiliated Hospital of Guangzhou University of TCM)The rats were fed by standard lab animal food (supported by the lab animal center of Guangdong). The rats, except the Normal group, were sensitized by Ovalbumin (OVA) to become asthmatic. The Group B, C, D and E rats were intraperitoneal injected with10%OVA10%Al (OH)3(aluminum hydroxide) mixed solution as sensitization,1ml each rat, on the1st day and8th day. Then, the 4groups rats were put into a proper air tight box(47×47×47cm) to be ultrasonic nebulized with2%OVA solution,30minutes each time,1time each day, These stimulations continued till the28th day.The AISB group was additionally treated since the17th day. The course of treatment for the AISB group consisted of0.4ml injection of its own blood into each acupoint, the bilateral Feishu (BL13) and Shenshu (BL23) alternately, with one injection every two days, for a total of six times. The NS group was just acupoint injected with normal saline. Its beginning moment, does, selection of acupoints, frequency, and the course was as the same as the AISB group. The DXM group was intraperitoneal injection with Dexamethasone solution, taking0.5mg/kg according to rats'each weight as the does. And its beginning moment, frequency and the course was as the same as the AISB group. All the clinical treatments were done30minutes before the ultrasonic nebulization. The weight (g) of rats were measured and recorded at less1time per week. On the29th day, the serum and lung tissues were collected from all the rats taken anesthesia with Chloral Hydrate. And the corpses of rats were packaged and abandoned following the rules of the animal lab.The tests of materials (serum and lung tissue) included five parts:The first part, the expression of asthmatic related cells (EOS, mastocyte, granulocyte, lymphocyte, et al.) and the condition of bronchus in rats'lung tissue slices were observed though the pathological test (H-E staining). The second part, the contents(pg/ml) of IL-4, IL-5, IFN-γ, IL-17and IL-10in rats'serum were recorded though enzyme-linked immunosorbent assay (ELISA) tests. The third part, the ratios of IL-4/IFN-γ and IL-5/IFN-γ contents were counted and recorded additionally. The expression levels(staining index values) of GATA3, T-bet, IL-17and FoxP3protein in rats'lung tissue slides were recorded though immunohistochemical (IHC)(SP staining) test and the computer photographic analysis. The fourth part, the expression levels (calibrated value, CV) of GATA3mRNA, T-bet mRNA, IL-17mRNA and FoxP3mRNA in rats'lung tissues were recorded though Realtime-polymerase chain reation (RT-PCR) tests. And the ratios of expression levels of GATA3mRNA/T-bet mRNA were counted and recorded additionally. The fifth part, the initial weight (g)(Wt1, measured on the1st day), last weight (g)(Wt4, measured on the29th day), the difference of the last and initial weight (g)(WtD, WtD=Wt4-Wt1) and the ratio of the last over initial weight (WtR, WtR=Wt4/Wt1) of rats were measured and recorded.After all the tests were finished, the records were made the database on the Excel (software) tables. Then the statistics work was done with the software (SPSS16.0and STATA10.0), as the size of test is0.05(α=0.05) and the significant one is0.01(α=0.01).ResultsPart Ⅰ. The pathological observation of the rats'lung tissue slices by HE staining:The Model rats:mucus plugs were found in bronchus, hyperplasia of cylindric cell on the epithelium mucosae, thickening of basilar membrane, severely aggregation of eosinophile, monocyte, lymphocyte and plasmocyte be found on the lamina propria, submucous layers and muscle layers.The AISB rats:mucus plugs were reduced in bronchus, no damage on the epithelium mucosae, no thickening of basilar membrane, light degree of aggregation of lymphocytes in bronchus walls, no significant eosinophilia, no hyperplasia of fibrous connective tissue or asthmatic inflammatic cells, comparing with the Model group.The differences of the Normal and Model rats showed us the copying of asthmatic rats was successful. The expression of asthmatic inflammation in the AISB and DXM rats, were significantly lower than that in the Model and NS rats.Part Ⅱ. The ELISA test of asthmatic inflammation cytokine in rats' serum:The mean level of serum IL-4of rats:Model>NS>AISB>DXM>Normal rats. The mean level of serum IL-5of rats:Model>NS>DXM>AISB>Normal rats. The mean level of serum IFN-y of rats:DXM>NS>AISB>Model> Normal rats. In the Model rats, the expression of serum IL-4, IL-5, and IFN-Y, were significantly higher than the Normal rats (P<0.01). In the AISB rats, the expression of serum IL-4and IL-5, were significantly lower than the Model rats (P<0.01), no differences with the Normal rats (P>0.05); In the AISB rats, the expression of serum IFN-Y, no differences with the Model rats (P>0.05), but was higher than the Normal rats (P<0.05). In the NS rats, the the expression of serum IL-4, IL-5, and IFN-γ, no differences with the Model rats (P>0.05). In the DXM rats, the expression of serum IL-4and IL-5, were significantly lower than the Model rats (P<0.01), no differences with the AISB rats (P>0.05); In the DXM rats, the expression of serum IFN-γ, was higher than the Model rats (P<0.05), no differences with the AISB rats(P>0.05).The mean ratio of serum IL-4/IFN-γ of rats:Model>NS>Normal>DXM>AISB rats. The mean ratio of serum IL-5/IFN-γ of rats:Model>DXM>NS>Normal>AISB rats. In the Model rats, both the ratio of serum IL-4/IFN-γ and the raito of serum IL-5/IFN-γ, were higher than the Normal rats (P <0.05). In the AISB rats, the ratio of serum IL-4/IFN-γ, was lower than the Model rats (P<0.05), no differences with the Normal rats (P>0.05); In the AISB rats, the ratio of serum IL-5/IFN-γ, was significantly lower than the Model rats (P<0.01), no differences with the Normal rats (P>0.05). In the NS rats, the ratio of serum IL-4/IFN-γ, was no differences with the Model rats (P>0.05); In the NS rats, the ratio of serum IL-5/IFN-γ, was lower than the Model rats (P<0.05). In the DXM rats, the ratio of serum IL-4/IFN-γ, was significantly lower than the Model rats (P<0.01); In the DXM rats, the ratio of serum IL-5/IFN-γ, no differences with the Model rats (P>0.05).The mean level of serum IL-17of rats:Model>NS>DXM>AISB>Normal rats. In the Model rats, the expression of serum IL-17, was significantly higher than the Normal rats (P<0.01). In the AISB rats, the expression of serum IL-17, was lower than the Model rats (P<0.05), no differences with the Normal rats (P>0.05). In the NS rats, the expression of serum IL-17, no differences with the Model rats (P>0.05), was higher than the Normal rats (P<0.05). In the DXM rats, the expression of serum IL-17, was lower than the Model rats (P<0.05), no differences with the AISB rats (P>0.05)The mean level of serum IL-10of rats:AISB>NS>Normal>DXM>Model rats. In the Model rats, the expression of serum IL-10, was lower than the Normal rats (P<0.05). In the AISB rats, the expression of serum IL-10, was significantly higher than the Model rats (P<0.01), no differences with the Normal rats (P>0.05). In the NS rats, the expression of serum IL-10, was higher than the Model rats (P<0.05), no differences with the AISB rats (P>0.05). In the DXM rats, the expression of serum IL-10, no differences with the Model rats (P>0.05), was lower than the AISB rats (P<0.05)Part Ⅲ. The immunohistochemical (IHC) test (SP staining) and the computer photographic analysis of asthmatic inflammation protein in rats'lung tissue slices. After the IHC SP staining, the expression of aim protein turned yellow brown on the rats' lung tissue slices. In the IHC observation of GATA3, we found there is low level expression on the Normal rats', high level expression on the Model and NS rats', moderate level expression on the AISB and DXM rats' In the IHC observation of T-bet, we found there is low level expression on the Normal and DXM rats', high level expression on the NS and AISB rats' In the IHC observation of IL-17, we found there is low level expression on the Normal rats', high level expression on the Model and NS rats', low to moderate level expression on the AISB and DXM rats'. In the IHC observation of FoxP3, we found there is low level expression on the Normal rats', low level expression on the Model and NS rats', high level expression on the AISB rats', low to moderate level expression on the DXM rats'According to the computer photographic analysis of GATA3mean expressed on the rats' slices:Model>NS>DXM>AISB>Normal rats. The GATA3expressed on the Model and NS rats', both were significantly higher than the Normal rats'(P<0.01). There was no differences between the Model and NS rats'(P>0.05). The GATA3expressed on the AISB and DXM rats', both were significantly lower than the Model rats'According to the computer photographic analysis of T-bet mean expressed on the rats' slices:NS>AISB>Normal>DXM>Model rats. There was no differences among the Model, DXM and Normal rats'(P>0.05). The T-bet expressed on the AISB and NS rats', both were significantly higher than the Model rats'(P<0.01), both were higher than the Normal rats'(P<0.05), both were higher than the DXM rats'. The T-bet expressed on the AISB, no differences with the NS rats'(P>0.05)According to the computer photographic analysis of IL-17mean expressed on the rats'slices:Model>NS>AISB>Normal>DXM rats. The IL-17expressed on the Model rats', was significantly higher than the Normal rats'(P<0.01). The IL-17expressed on the AISB rats', was significantly lower than the Model rats'(P<0.01), no differences with the Normal rats'(P>0.05). The IL-17expressed on the NS rats', no differences with the Model rats'(P>0.05), lower than the AISB rats'(P<0.05). The IL-17expressed on the DXM rats', lower than the Model rats'(P<0.05), no differences with the AISB rats'(P>0.05)According to the computer photographic analysis of FoxP3mean expressed on the rats'slices:AISB>DXM>NS>Model>Normal rats. The FoxP3expressed on the Model rats', no differences with the Normal rats'(P>0.05). The FoxP3expressed on the AISB rats', higher than the Model rats'(P<0.05), was significantly higher than the Normal rats'(P<0.01). The FoxP3expressed on the NS rats', no differences with the Model rats'(P>0.05), lower than the AISB rats'(P<0.05). The FoxP3expressed on the DXM rats', no differences with the Model rats'(P>0.05), no differences with the AISB rats'(P>0.05).Part IV. The Realtime-polymerase chain reation test (RT-PCR) of the expression levels (calibrated value, CV) of GATA3mRNA, T-bet mRNA, IL-17mRNA and FoxP3mRNA in rats'lung tissues. And the ratios of expression levels of GATA3mRNA/T-bet mRNA (G/T) were counted and recorded additionally.The mean CV of the GATA3mRNA on the rats':Model>NS>DXM>AISB> Normal rats'. The GATA3mRNA expressed in the Model and NS rats', both were significantly higher than the Normal rats'(P<0.01). The GATA3mRNA expressed in the AISB, NS and DXM rats', were significantly lower than the Model rats'(P<0.01). The GATA3mRNA expressed in the AISB rats', lower than the NS rats'(P<0.05)The mean CV of the T-bet mRNA on the rats':Normal>AISB>DXM>NS> Model rats'. The T-bet mRNA expressed in the Model and NS rats', both were significantly lower than the Normal rats'(P<0.01). The T-bet mRNA expressed in the AISB rats', was significantly higher than the Model rats'(P<0.01), was higher than the NS rats'(P<0.05), no differences with the DXM rats'(P>0.05)The mean G/T ratio in the rats':Model>NS>DXM>AISB>Normal rats' The G/T in the Model rats', was significantly higher than the Normal rats'(P<0.01). The G/T in the AISB and DXM rats', were significantly lower than the Model rats'(P<0.01). The G/T in the AISB rats', was lower than the DXM rats'(P<0.05), no differences with the DXM rats'(P>0.05)The mean CV of the IL-17mRNA on the rats':Model>NS>DXM>AISB> Normal rats'. The IL-17mRNA expressed in the Model rats', was significantly higher than the Normal rats'(P<0.01). The IL-17mRNA expressed in the AISB rats', was significantly lower than the Model rats'(P<0.01), was significantly higher than the Normal rats'(P<0.01). The IL-17mRNA expressed in the NS rats', no differences with the Model rats'(P>0.05), was significantly higher than the AISB rats'(P<0.01). The IL-17mRNA expressed in the DXM rats', was significantly lower than the Model rats'(P<0.01), no differences with the AISB rats'(P>0.05)The mean CV of the FoxP3mRNA on the rats':AISB>Normal>NS>DXM> Model rats'. The FoxP3mRNA expressed in the Model rats', was significantly lower than the Normal rats'(P<0.01). The FoxP3mRNA expressed in the AISB rats', was significantly higher than the Model rats'(P<0.01), was significantly higher than the Normal rats'(P<0.01). The FoxP3mRNA expressed in the NS rats', was higher than the Model rats'(P<0.01), was lower than the AISB rats'(P<0.05). The FoxP3mRNA expressed in the DXM rats', was significantly higher than the Model rats'(P<0.01), was lower than the AISB rats'(P<0.05)Part V. Weigh the rats and compare the differencesThe initial weight (g)(Wt1) of all rats:the mean and s was132.458+8.282g, the minimum value was110g, and the maximal value was153g. The Wtl among the5groups was in balance.The mean last weight (g)(Wt4) of the rats:Model>AISB>Normal>NS>DXM rats. There was no differences among the Model, Normal, AISB and the NS rats'Wt4(P>0.05). The Wt4of the DXM rats, was significantly lower than the Model rats (P<0.01), was significantly lower than the AISB rats (P<0.01), was significantly lower than the NS rats (P<0.01), was significantly lower than the Normal rats (P<0.01)The mean WtR (WtR=Wt4/Wt1) of the rats:Model>Normal>AISB>NS>DXM rats. There was no differences among the Model, Normal, AISB and the NS rats'WtR (P>0.05). The WtR of the DXM rats, was significantly lower than the Model rats (P<0.01), was significantly lower than the AISB rats (P<0.01), was significantly lower than the NS rats (P<0.01), was significantly lower than the Normal rats (P<0.01)Conelusion1. The Acupoint injection with self-blood (AISB) is effective in reducing the severity of asthmatic inflammation in model rats, both in the peripheral circulation and local lung, reducing the risk factors (IL-4, IL-5, GATA3, IL-17and related mRNA), and increasing the protective factors (IFN-γ, IL-10, T-bet, FoxP3and related mRNA). What is more, the AISB could step down the progression and improve the recovery in asthma.2. The curing effects of acupoint injection with normal saline on asthma is weak, less effective than the AISB.3. The Dexamethasone treatment worked as effective as AISB in reducing the risk factors in asthmatic rats, but much less effective than the AISB in the increasing the protective factors. What is opposite, it showed that Dexamethasone negatively influencing the weight growing of asthmatic rats. Than the weight growing of asthmatic rats keep on normal speed both in AISB and NS group. That is interesting.