Experimental Study On Anti - Fatigue Mechanism Of Acupuncture At Spleen - Shao Point Combined With Ginsenoside Rg3 Based On "Spleen Qi And Qi"

Purpose: The purpose of the study is to discuss the mechanism of “Synergistic interaction of acupuncture and administration”, based on which acupuncture at BL20 and administration of ginsenoside Rg3 will replenish qi and invigorate the spleen, thus perform anti-fatigue function. The study was conducted with following methods: In Chronic Fatigue model rats, High Performance Liquid Chromatography（HPLC） was applied to observe the effect of Acupuncture at BL20 on ginsenoside Rg3 distribution and metabolism in organs at different time phase; Optical Microscope was utilized to view T-Lymphocyte Transformation Ratio in rat serum; Flow Cytometry Assay was made use of to determine the proportion of T-Cell Subsets: CD₃⁺T-cells, CD₄⁺T-cells, and CD₈⁺T-cells, as well as the ratio of CD₄⁺/CD₈⁺ in spleen of rats; ELISA was employed to examine the content variation of IFN-γ and IL-4 in serum; RT-PCR was adopted to test the Expression Level of IFN-γ and IL-4 m RNA in spleen;and Western Blot was exploited to measure IFN-γ and IL-4 protein expression level in spleen. Material and methods: Thesis One:116 Male Wistar Rats was randomized into 4 groups: Blank group（33 animals）, Model group（33 animals）, Blank and Acupuncture group（25 animals）, Model and Acupuncture group（25 animals）. Model replication process was performed by forced swimming in cold water and chronic bandage. Rat began to swim at 8:00 a.m. in plastic bucket which is 90 cm in height and 60 cm in diameter, with water 40-45 cm in depth at 15±2℃Forced swimming stops when the coordination in swimming disappeared and it last longer than 10 s that the nose-tip was submerged, the body sank and then resurfaced. During swimming, avoid the huddling of rats for self-rescue or sinking out of exhaustion. The animals were pulled out and wiped with cloth. They were dried with hair dryer, after which were settle back to their rat cage with corresponding environment. Every 3:00 p.m., the rats were put into homemade bandage tubes for 1h, and then taken back to their cages.Time for model duplication was 21 d. Following 21 d of modeling, 8 rats were randomly taken from Blank group and Model group, respectively. Whole blood 2ml from post-glomus venous plexus were taken, and the serum was segregated for lactic acid content. Modeling acceptation depended on the assessment results on the variation of body weight and serum lactic acid content.In Blank and Acupuncture group, together with Model and Acupuncture group, modeling replication success was followed by the treatment of acupuncture at BL20. After needling, the needle handle was connected to the Electro-acupuncture treatment machine with a voltage of 4.5 V and condensation-rarefaction wave at a frequency of 4/20 Hz, and hold the strength as local skin and muscle are slight trembling. The needles were retained for 20 min. Treatment was performed once daily for 7 days. Upon the completion of acupuncture on 7th day, the rats were intravenously injected at tail with ginsenoside Rg3. Five, 30, 60 and 120 min post-dose, 2ml blood was captured from eye orbit of the rats, 5 animals respectively in each group, into heparinization centrifuge tube, and centrifuged for 10 min at 3000 rpm. Following plasma separation, HPLC was applied to observe ginsenoside Rg3 content during each period in rat plasma of each group.The 20 remaining rats were sacrificed, 5 animals from a group each time, at 0.5, 1, 2 and 4h, respectively, for tissues of heart, liver, spleen, lung and kidney, 0.5g of which were added with methanol 1ml. Tissue homogenate was prepared, vibrated for 10min; Ultrasonic method was applied for 10 min, and centrifugation method was applied for 10 min at 4000 rpm. Supernatant was collected for HPLC to determine ginsenoside Rg3 content in rat heart, liver, spleen, lung and kidney during each time period.Thesis Two: Forty male Wistar Rats were randomized into Blank Control group, Model group, Ginsenoside group, Acupuncture group, and Ginsenoside Combined with Acupuncture group, 8 animals in each. Model replication process was performed by forced swimming in cold water and chronic bandage. Rat began to swim at 8:00 a.m. in plastic bucket which is 90 cm in height and 60 cm in diameter, with water 40-45 cm in depth at 15±2℃. Forced swimming stops when the coordination in swimming disappeared and it last longer than 10 s that the nose-tip was submerged, the body sank and then resurfaced. During swimming, avoid the huddling of rats for self-rescue or sinking out of exhaustion. The animals were pulled out and wiped with cloth. They were dried with hair dryer, after which were settle back to their rat cage with corresponding environment. Every 3:00 p.m., the rats were put into homemade bandage tubes for 1h, and then taken back to their cages.Time for model duplication was 21 d. After the success of model replication process, in Acupuncture group rats were treated with acupuncture at “BL20”, after needling, the needle handle was connected to the Electro-acupuncture treatment machine with a voltage of 4.5 V and condensation-rarefaction wave at a frequency of 4/20 Hz, and hold the strength as local skin and muscle are slight trembling. The needles were retained for 20min; rats were dosed with ginsenoside Rg3, intravenous injection at tail, in Ginsenoside group; rats were dosed with ginsenoside Rg3 by intravenous injection at tail, and then acupunctured at “BL20” in Ginsenoside Combined with Acupuncture group 1/d for a 7d continuous treatment. One hour after last dosing and acupuncture, rats in each group were docked for blood to make blood smear. Optical microscope was utilized to observe T-Lymphocyte transformation ratio in rat blood serum; Following abdominal aortic method, spleen was taken for spleen cell suspension. Flow Cytometry Assay was applied to determine the proportion of T-Cell Subsets: CD₃⁺T-cell, CD₄⁺T-cell and CD₈⁺T-cell, as well as the ratio of CD₄⁺/CD₈⁺ in rat spleen.Thesis Three: Forty male Wistar Rats were randomized into Blank Control group, Model group, Ginsenoside group, Acupuncture group, and Ginsenoside Combined with Acupuncture group, 8 animals in each. After the completion of rat model replication and intervene in every group, the rats were intraperitoneally injected with 10% Chloral Hydrate at 3ml/kg body weight for Anesthesia. Abdominal aorta puncture was applied to capture whole blood. Supernatant liquor was stored in refrigerator at-80℃, until the content of IFN-γ and IL-4 in serum was examined in Elisa Experiment. Then, rat spleen tissues were collected and divided into two, after which RT-PCR was adopted to test the expression level of IFN-γ and IL-4 m RNA in spleen, and Western Blot to measure the protein expression level of IFN-γ and IL-4. Results:Thesis One:1 Model Evaluation: The difference of rat body weight in both groups is of no statistical significant（p>0.05）. After modeling, the body weight has significantly reduced（p<0.01）, and lactic acid content has significantly risen（p<0.01） in comparison to Blank group, which indicates modeling replication is success.2 Ginsenoside Rg3 Distribution in Blood Plasma of Rats in Each Group: In comparison with Blank group, the content of ginsenoside Rg3 in blood plasma has significantly decreased at 60 min and 120 min in Model group, and significantly increased at 60 min and 120 min in Blank Acupuncture group（P<0.05）; In comparison with Model group, content of ginsenoside Rg3 has significantly increased at 60 min and 120 min in Model Acupuncture group and Blank Acupuncture group（P<0.05）. Ginsenoside Rg3 content in Blood Plasma reaches the peak at 5min, when it begins to decrease.3 Ginsenoside Rg3 Distribution in Liver of Rats in Each Group: In comparison with concurrent Blank Control group, the content of ginsenoside Rg3 has significantly decreased at 1h, 2h and 4h in Model group（P<0.05）, and significantly increased at 1h, 2h and 4h in Blank Acupuncture group（P<0.05）, while no significant variation occurred in Model Acupuncture group; In comparison with Model group, content of ginsenoside Rg3 has significantly increased at 1 h, 2h and 4h in Model Acupuncture group and Blank Acupuncture group（P<0.05）. Ginsenoside Rg3 content in kidney reaches the peak at 1h, when it begins to decrease.4 Ginsenoside Rg3 Distribution in Heart of Rats in Each Group: In comparison with Blank Control group, the content of ginsenoside Rg3 has significantly decreased at 0.5h and 1h in Model group（P<0.05）, and significantly increased at 0.5h and 1h in Blank Acupuncture group（P<0.05）; In comparison with Model group, content of ginsenoside Rg3 has significantly increased at 0.5h and 1h in Model Acupuncture group and Blank Acupuncture group（P<0.05）. Ginsenoside Rg3 content in rat heart tissue reaches the peak at 0.5h, when it begins to decrease. No ginsenoside Rg3 was detected at 4h in rat heart tissue in any of the 4 groups.5 Ginsenoside Rg3 Distribution in spleen of Rats in Each Group: In comparison with Blank Control group, the content of ginsenoside Rg3 has significantly decreased at all timepoints, except at 4h, in Model group（P<0.05）, and significantly increased at all timepoints, except at 4h, in Blank Acupuncture group（P<0.05）; In comparison with Model group, content of ginsenoside Rg3 has significantly increased at all time-points, except at 4h, in Model Acupuncture group and Blank Acupuncture group（P<0.05）. Ginsenoside Rg3 content in spleen reaches the peak at 0.5h, when it begins to decrease.6 Ginsenoside Rg3 Distribution in Lung of Rats in Each Group In comparison with Blank Control group, the content of ginsenoside Rg3 has decreased at each time-point in Model group, increased in Blank Acupuncture group, but the difference is not statistically significant（P>0.05）; In comparison with Model group, content of ginsenoside Rg3 has significantly increased at 1h and 2h Blank Acupuncture group（P<0.05）, while the content of ginsenoside Rg3 has increased at all time-points with no statistical significance（P>0.05）. No ginsenoside Rg3 was detected at 4h in lung tissue of rats in any group of the four.7 Ginsenoside Rg3 Distribution in Kidney of Rats in Each Group: In comparison with Blank Control group, the content of ginsenoside Rg3 has significantly decreased at 2h and 4h in Model group（P<0.05）, and significantly increased at 2h and 4h in Blank Acupuncture group（P<0.05）; In comparison with Model group, content of ginsenoside Rg3 has significantly increased at 2h and 4h in Model Acupuncture group and Blank Acupuncture group（P<0.05）. Ginsenoside Rg3 content in kidney reaches the peak at 1h, when it begins to decrease.Thesis Two:1 Results of Lymphocyte Transformation Ratio of Rats in Each Group: In comparison with Blank Control group, the lymphocyte transformation ratio of the rats in each group has significantly decreased（P<0.01）; In comparison with Model group, lymphocyte transformation ratio has significantly increased in Acupuncture group, Ginsenoside group and Ginsenoside Combined with Acupuncture group（P<0.01）; In comparison with Ginsenoside Combined with Acupuncture group, the lymphocyte transformation ratio has significantly decreased in Acupuncture group and Ginsenoside group（P<0.05）; while the difference between Ginsenoside group and Acupuncture group is not of statistical significant（P>0.05）.Normal Lymphocyte appears to be perfect sphere; cell nucleus appears to be purple perfect sphere; endochylema is little and appears to be nattier blue, in shape of crescent, locating at the edge of the cell. When transformed, lymphoblast enlarges, and the cell nucleus becomes bigger. Nuclous appears occasionally. The shape is irregular. Endochylema becomes more. In addition, there are vacuoles incidentally.2 Results of Flow Cytometry Assay:Results of CD₃⁺ T-cell Proportions: In comparison with Blank Control group, the proportion of CD₃⁺ T-cell has significantly decreased in Model group（P<0.01） and Ginsenoside group（P<0.05）; In comparison with Model group, the proportion of CD₃⁺ T-cell has significantly increased in Model group（P<0.01） and Ginsenoside Combined with Acupuncture group（P<0.05）; while the difference between Ginsenoside group and Acupuncture group is not of statistical significant（P>0.05）.Results of CD₄⁺ T-cell Proportion: In comparison with Blank Control groups, the proportion of CD₄⁺ T-cell has increased, and the difference is statistical significant（P<0.05）; In comparison with Model group, the proportion of CD₄⁺ T-cell has decreased in Ginsenoside Combined with Acupuncture group, and the difference is statistical significant（P<0.05）.Results of CD₈⁺ T-cell Proportion: In comparison with Blank Control group, the proportion of rat CD₈⁺T-cell has significantly decreased in each group（P<0.01）; In comparison with Model group, the proportion of CD₈⁺ T-cell has significantly increased in Acupuncture group, Ginsenoside group and Ginsenoside Combined with Acupuncture group（P<0.01）. In comparison with Ginsenoside Combined with Acupuncture group, the proportion of CD₈⁺ T-cell has significantly decreased（P<0.01）.The results of CD₄⁺/CD₈⁺: In comparison with Blank Control group, the ratio of CD₄⁺/CD₈⁺ has significantly increased in Model group and Ginsenoside group（P<0.01）; In comparison with Model group, the ratio of CD₄⁺/CD₈⁺ has significantly decreased in Acupuncture group, Ginsenoside group and Ginsenoside Combined with Acupuncture group（P<0.01）. In comparison with Ginsenoside Combined with Acupuncture group, the ratio of CD₄⁺/CD₈⁺ has significantly increased in Ginsenoside group（P<0.05）.Thesis Three:1 the results of content of IFN-γ and IL-4 in Serum of the Rats in Each GroupIn comparison with Blank Control group, the contents of IL-4 in serum of the rats in each group have significantly decreased（P<0.01）; In comparison with Model group, the contents of IL-4 m RNA have significantly increased within the rats in Acupuncture group, Ginsenoside group and Ginsenoside Combined with Acupuncture group（P<0.01）; In comparison with Ginsenoside Combined with Acupuncture group, the contents of IL-4 m RNA in Acupuncture group and Ginsenoside group have significantly decreased（P<0.01）; while the difference between Ginsenoside group and Acupuncture group is not of statistical significant（P>0.05）.In comparison with Blank Control group, the contents of IL-4 in serum of the rats in each group have significantly increased（P<0.01）; In comparison with Model group, the contents of IL-4 m RNA have significantly decreased within the rats in Acupuncture group, Ginsenoside group and Ginsenoside Combined with Acupuncture group（P<0.01）; In comparison with Ginsenoside Combined with Acupuncture group, the contents of IL-4 m RNA in Acupuncture group and Ginsenoside group have significantly increased（P<0.01）; while the difference between Ginsenoside group and Acupuncture group is not of statistical significant（P>0.05）.2 Results of Protein Expression of IFN-γ and IL-4 in the Spleen of Rats in Each Group:In comparison with Blank Control group, the expression levels of IFN-γ within the rats in each group have significantly decreased（P<0.01）; In comparison with Model group, the expression levels of IFN-γ have significantly increased within the rats in Acupuncture group, Ginsenoside group and Ginsenoside Combined with Acupuncture group（P<0.01）; In comparison with Ginsenoside Combined with Acupuncture group, the expression levels of IFN-γ in Acupuncture group and Ginsenoside group have significantly decreased（P<0.05）; while the difference between Ginsenoside group and Acupuncture group is not of statistical significant（P>0.05）.In comparison with Blank Control group, the expression levels of IL-4 within the rats in each group have increased, and the difference was significant（P<0.01）; In comparison with Model group, the expression levels of IL-4 have decreased within the rats in Acupuncture group, Ginsenoside group and Ginsenoside Combined with Acupuncture group, and the difference was significant（P<0.01）; In comparison with Ginsenoside Combined with Acupuncture group, the expression levels of IL-4 in Acupuncture group and Ginsenoside group have increased, and the difference is significant（P<0.05）; while the difference between Ginsenoside group and Acupuncture group was not of statistical significant（P>0.05）.3 Expression Results of IFN-γ and IL-4 m RNA in the Spleen of Rats in Each Group:In comparison with Blank Control group, the expression levels of IFN-γ m RNA within the rats in each group have significantly decreased（P<0.01）; In comparison with Model group, the expression levels of IFN-γ m RNA have significantly increased within the rats in Acupuncture group, Ginsenoside group and Ginsenoside Combined with Acupuncture group（P<0.01）; In comparison with Ginsenoside Combined with Acupuncture group, the expression levels of IFN-γ m RNA in Acupuncture group and Ginsenoside group have significantly decreased（P<0.05）; while the difference between Ginsenoside group and Acupuncture group is not of statistical significant（P>0.05）.In comparison with Blank Control group, the expression levels of IL-4 m RNA within the rats in each group have significantly increased（P<0.01）; In comparison with Model group, the expression levels of IL-4 m RNA have significantly decreased within the rats in Acupuncture group, Ginsenoside group and Ginsenoside Combined with Acupuncture group（P<0.01）; In comparison with Ginsenoside Combined with Acupuncture group, the expression levels of IL-4 m RNA in Acupuncture group and Ginsenoside group have significantly increased（P<0.05）; while the difference between Ginsenoside group and Acupuncture group iss not of statistical significant（P>0.05）. Conclusion: 1.Acupuncture at “BL20” may increase the concentration of ginsenoside Rg3 within the blood, strengthen the distribution in the five zang viscera, and prolong the residence time in vivo, which may be the material basis of acupuncture combination of drugs for enhancing the curative effect. 2.Acupuncture on “BL20” and intravenous injection of ginsenoside Rg3 may perform a regulation for equilibrium of Th1/Th2 and T-Lymphocyte Subsets in Fatigue Model Rats, which may be one of the mechanisms of anti-fatigue function. 3.Both acupuncture at “BL20” and intravenous injection of ginsenoside Rg3 have the function of anti-fatigue. The combination may perform a synergistic interaction.