| Purpose: In this experiment,spontaneously hypertensive rats were used as animal models.Under the theoretical guidance of "treating from the spleen",the mechanism of warming acupuncture and moxibustion at Zusanli point to reduce blood pressure in spontaneously hypertensive rats was explored.The signaling pathway(TLR4 / My D88 / NF-?B)mediates the downstream inflammatory response,thereby exploring the anti-inflammatory mechanism and endothelial cell protection mechanism of warm acupuncture on spontaneously hypertensive rats,providing a reliable basis for the clinical prevention and treatment of hypertension in traditional Chinese medicine Theoretical basis.Materials and methods: Ten male Kyoto species(WKY)rats were included in the blank group,and 40 male spontaneously hypertensive rats(SHR)were randomly divided into the model group,warm acupuncture group,acupuncture group and moxa according to the random number table method Moxibustion group,10 animals in each group.The conventional feed was fed adaptively for one week,and the intelligent non-invasive blood pressure meter was used to measure the blood pressure of the tail artery of each group of rats,and the average value was measured 3 times.To start the experiment,the rats in the normal group and the model group were fixed for 20 minutes,once a day,without acupuncture intervention;the rats in the warm acupuncture group were given acupuncture and moxibustion on both sides of Zusanli for 20 minutes;Acupoint 20min;Moxibustion group rats Moxibustion bilateral Zusanli point 20 min,once a day,continuous treatment for 15 days,at the same time measuring the first,3,5,7,9,11,13,15,17 after the start of the experiment,19,21 days 9o'clock to 11 o'clock in the morning,the blood pressure of each group of rats,measured three times to take the average,enter Excel and wait for statistics.After the treatment,the rats in each group were anesthetized with chloral hydrate(10%),fixed,depilated and disinfected,opened the abdomen along the midline of the abdomen,exposed the abdominal aorta and quickly collected blood,centrifuged the blood,and took the supernatant into sterile Store in EP tube for later use,and detect the content of serum inflammatory factors IL-6,IFN-? and TNF-? by ELISA;the content of serum immune indexes Ig A,Ig M and Ig G;the content of vasoconstrictor factors Ang-?,ET-1 and VEGF.Take a complete 1 cm section of abdominalaorta,rinse and fix with 4% paraformaldehyde solution,repair,embed,and slice with HE staining method,and observe the pathological morphological changes of abdominal aorta wall under fluorescence microscope.Explore the abdomen,quickly take 50 mg of spleen tissue into an EP tube and freeze it for later use.HE staining will fix,embed,and prepare the fixed spleen tissue of the rat,and observe the splenic histopathological changes and inflammatory cells under a fluorescent microscope.Infiltration state;take 50 mg of spleen tissue,grind,centrifuge,draw supernatant,extract protein of spleen tissue by Western Blot method,use BCA detection method to determine protein concentration,calculate gray value of gray band image by Image J software,quantitative analysis Group TLR4,My D88,NF-?B p65 protein expression and NF-?B p65 protein phosphorylation level;Take 100 mg of spleen tissue,extract the m RNA of rat spleen tissue by Trizol method,use q PCR method,through reverse transcription reaction,PCR amplification reaction,export q PCR data and calculate and analyze the expression level of TLR4,My D88,NF-?B p65 m RNA.Statistical data were analyzed using SPSS 22.0 software.The experimental results were expressed as mean standard deviation(x s);ne-way analysis of variance(one-way AN VA)was used to compare data differences between groups,with P 0.05 indicating statistical difference.Results:1.Changes in blood pressure of rats in each group before and after treatment: Blood pressure in the warm acupuncture group and acupuncture group began to gradually decrease on the second day of the experiment,and blood pressure began to decrease significantly on the seventh day of the experiment.The decrease in the warm acupuncture group was greater than that of the acupuncture group;on the 15 th day of the experiment At the end of the experimental intervention,the blood pressure decreased significantly in the warm acupuncture group,the acupuncture group,and the moxibustion group.After the experimental intervention,the blood pressure continued to be measured until the 21 st day and it was found that the blood pressure in the warm acupuncture group and the moxibustion group was relatively stable without significant fluctuations.In the acupuncture group,the model group and the normal group,the blood pressure all fluctuated to varying degrees,and the blood pressure of the acupuncture group increased significantly.2.The distribution of lymph sheaths and lymphocytes around the spleen artery in each group of rats: The area of ??the white pulp in the normal group and the warm acupuncture group is relatively large,and the lymph sheath tissue around the artery is thick.The lymph nodes inside the sheath and the distribution outside the sheath can be clearly observed.Large and dense lymphocytes;while the area of ??the white pulp and spleen nodules of the model group,acupuncture group and moxibustion group has been reduced to varying degrees,the lymphatic sheaths around the arteries have been significantly reduced,and the lymphocytes around the sheaths are also scarcely distributed.The model group shrinks most obviously.3.Analysis of serum inflammatory factors IL-6,IFN-?,TNF-? in each group of rats:Compared with the normal group,the levels of IL-6 and TNF-? in the model group and moxibustion group were significantly increased,and the content of IFN-? Significantly decreased;TNF-? content increased significantly in warm acupuncture group and acupuncture group,and IFN-? content decreased significantly.Compared with the model group,the content of IL-6 and TNF-? in the warm acupuncture group was significantly reduced and the content of IFN-? was significantly increased;the content of TNF-? in the acupuncture group was significantly reduced,and the content of IFN-? in the moxibustion group was significantly increased.Compared with the warm acupuncture group,the IFN-? content in the acupuncture group was significantly reduced;the IL-6 and TNF-? levels in the moxibustion group were significantly increased,and the IFN-? content was significantly reduced.Compared with the acupuncture group,the TNF-? content in the moxibustion group was significantly increased.The above differences are statistically significant(P 0.05).4.Abnormal blood pressure and immune inflammation are closely related and interact: the systolic blood pressure and diastolic blood pressure are positively correlated with IL-6 and TNF-? content,and negatively correlated with IFN-? content,indicating that with the With the increase of blood pressure in rats,the content of IL-6 and TNF-? in the serum also increase,which accelerates the expression of serum inflammatory factors,while the content of IFN-? decreases accordingly.5.Toll-like receptor signaling pathway(TLR4 / My D88 / NF-?B)protein expression of various signal molecules and NF-?B p65 phosphorylation levels: compared with the normal group,model group,acupuncture group TLR4,My D88,NF-?B p65 protein expression andNF-?B p65 protein phosphorylation level were significantly increased.nly TLR4 protein expression and NF-?B p65 protein phosphorylation level were significantly increased in warm acupuncture group,while TLR4,NF-?B p65 protein expression and NF-?B p65 protein phosphorylation level was significantly increased;compared with the model group,TLR4,My D88,NF-?B p65 protein expression and NF-?B p65 protein phosphorylation level were significantly reduced in the warm acupuncture group;compared with the warm acupuncture group,the acupuncture group TLR4,My D88,NF-?B p65 protein expression and NF-?B p65 protein phosphorylation level in the thorn group were significantly increased,while TLR4,NF-?B p65 protein expression and NF-?B p65 protein phosphorylation level in the moxibustion group were significantly increased.The above differences are statistically significant(P 0.05).6.Gene expression of each signal molecule in the Toll-like receptor signaling pathway(TLR4/ My D88 / NF-?B): Compared with the normal group,the expression levels of TLR4,My D88 and NF-?B p65 m RNA in the spleen tissues of the model group,acupuncture group and moxibustion group were significantly increased,and the expression levels of TLR4 and My D88 in the warm acupuncture group were significantly increased;compared with the model group The expression levels of TLR4,My D88 and NF-?B p65 m RNA in the spleen tissue of the warm acupuncture and moxibustion group were significantly reduced,the expression levels of TLR4 in the acupuncture group were reduced,and the expression levels of TLR4 and My D88 m RNA in the moxibustion group were reduced;TLR4,My D88 and NF-?B p65 m RNA expression levels in the spleen tissues of rats in the acupuncture and moxibustion groups were significantly increased.The above differences are statistically significant(P 0.05).7.Analysis of HE staining results of abdominal aorta wall in each group: the aorta wall in the normal group is smooth and complete,the cells in each layer are arranged in an orderly manner,the endothelial cells have not seen shedding and proliferation,and the median smooth muscle cells and elastic fibers are closely arranged in an orderly manner;In the model group,the intima of the abdominal aorta wall was significantly hyperplastic,thickened,and raised,and endothelial cells were detached.The median smooth muscle layer was disorderly arranged,with hyperplasia and hypertrophy,and deformation and atrophy appeared in someareas.The elastic fiber spacing was uneven.There are ruptures and wrinkles,the outer membrane cells are not flat,and occasionally shedding;the inner membrane of the abdominal aorta wall in the warm acupuncture group is slightly rough,a little endothelial cells fall off,and the median smooth muscle cells are occasionally bent,wrinkled,and the elastic fiber gap is roughly The aortic wall of the rats in the acupuncture group was rough and uneven,and the endothelial cells were detached.The median smooth muscle cells proliferated,enlarged,and arranged disorderly.The elastic fibers were wrinkled and deformed,and the gaps were uneven.Part of the adventitia showed protrusions,deformation,and cell shedding;in the moxibustion group,the intimal folds and deformation of the abdominal aorta wall,and the endothelium Cells were detached.Part of the median smooth muscle cells atrophied and decreased,the arrangement was uneven,the elastic fiber gap was uneven,the outer membrane was not smooth,and occasionally hyperplasia.8.Comparison of serum immunological indicators in each group: Compared with the normal group,the serum Ig A and Ig G contents of the model group and moxibustion group were significantly reduced,the serum Ig M was significantly increased,and the serum Ig A content of the acupuncture group was significantly reduced;compared with the model group,Compared with the warm acupuncture group,the serum Ig A content of the acupuncture group was significantly reduced,and the serum Ig A and Ig G contents of the moxibustion group were significantly decreased.Compared with the group,the serum Ig G content in the moxibustion group was significantly reduced.The above differences are statistically significant(P 0.05).9.Comparison of serum Ang?,ET-1,and VEGF levels in each group: Compared with the normal group,the serum Ang?,ET-1,and VEGF levels in the model group and moxibustion group were significantly increased,and the serum Ang? and ET-1 levels in the acupuncture group were significantly increased.Compared with the model group,the contents of Ang?,ET-1,and VEGF in the warm acupuncture group were significantly reduced,the contents of ET-1 and VEGF in the acupuncture group were significantly reduced,and the contents of ET-1 in the moxibustion group were significantly reduced.The content of ET-1 in the acupuncture group increased significantly,while the content of Ang?,ET-1,and VEGF in the moxibustion group increased significantly.The above differences are statistically significant(P 0.05).Conclusion:1 Warm acupuncture and moxibustion at Zusanli can significantly reduce blood pressure in spontaneously hypertensive rats,and the effect is stable and lasting.2 There is a close relationship and mutual influence between abnormal blood pressure and immune inflammation status;warm acupuncture and moxibustion at Zusanli can significantly improve the inflammation status in spontaneously hypertensive rats.3 Warm acupuncture and moxibustion at Zusanli can regulate signal transduction by inhibiting the expression of TLR4,My D88,NF-?B p65 protein and m RNA in the Toll-like receptor signaling pathway of rat spleen tissue.4 Warm acupuncture and moxibustion at Zusanli can reduce the inflammatory state of serum and improve the anti-inflammatory ability of serum by regulating TLR4 / My D88 / NF-?B signaling pathway,and benign regulation of vasomotor function,thereby reducing the degree of inflammatory injury and exerting protection for endothelial cells effect. |
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