Study On The Mechanism Of Heat-sensitive Moxibustion And Its Different Courses Of Treatment For Osteoporosis Based On Connective Tissue And Nervous System

Objective:1.The morphological changes of connective tissue and spinal cord around acupoints were studied by heat-sensitive moxibustion at“Mingmen”acupoint,and further discussed by different duration of treatment.2.The expression of ERK1/2 and P38 proteins in superficial fascia and spinal cord under different time of heat-sensitive moxibustion was observed by SP method.The expression of ERK1/2 and P38 proteins in rat muscles under different time of heat-sensitive moxibustion was semi-quantitatively observed by Western-Blot method to determine the best course time of heat-sensitive moxibustion.3.By comparing the results of“Mingmen”point and“Zusanli”point in the treatment of osteoporosis with heat-sensitive moxibustion,the effect of different heat-sensitive acupoints on the therapeutic mechanism of heat-sensitive moxibustion was discussed.4.The concentration of phosphorus,calcium ions and alkaline phosphatase in rat serum was measured,and the concentration difference before and after 14 days of heat-sensitive moxibustion was calculated to explore the effect of heat-sensitive moxibustion in vivo from the biochemical perspective.5.Through the above steps to explore the mechanism of therapeutic effect of heat-sensitive moxibustion.Methods:1.Osteoporosis model of rats by OVX method:Female SD rats with clean grade SD were randomly divided into groups.After skin preparation and anesthesia,the ovaries were removed at the same time to make a rat model of osteoporosis.After modeling,vaginal secretions were taken for rayon staining,and the changes in the number of vaginal epithelial cells and white blood cells were observed to prove the success of castration.2.Observe the morphological changes of superficial fascia and spinal cord by heat-sensitive moxibustion with HE staining:Osteoporosis rats were randomly divided into five groups,achieved“Mingmen”as the central area near 0.5-1.5cm subcutaneous superficial fascia,in addition,a gray-white cylindrical spinal cord was exposed by cutting along the pedicle of the vertebral arch with a bone-biting forceps.The spinal cord was stripped layer by layer with forceps.After washing,fixation,dehydration and waxing,two pieces ofparaffin-proof section for staining.3.SP method was used to observe the expression of ERK1/2 and P38 proteins in the superficial fascia and spinal cord:After the slices were prepared in accordance with the above steps,immunohistochemical method(SP method)was used for staining,and Image pro plus6.0 was used to calculate the average optical density value(MD)of the image.Then,GraphPadPrism6.0 was used to analyze the statistical results.If P<0.05 or P<0.01,the statistical significance was significant.4.Western blot method was used to detect the expression of ERK1/2and P38 protein in muscles near“Mingmen”point:KeyGen whole protein extraction is utilized to extract groups“Mingmen”area tissue protein and to measure protein concentration,follow the protein denaturation,glue,sample,plastic cutting,transfer film,sealing,steps draw five groups such as color gray stripe,the Alpha View of software to determine stripe grey value,and use the One-Way ANOVA analysis of variance to detect whether there is statistical significance between groups.5.Phosphomolybdate method,microenzyme labeling method and microplate method were respectively used to measure the concentrations of phosphorus,calcium ions and alkaline phosphatase in rat serum before and after 14 days of heat-sensitive sensitive moxibustion.The data were expressed in~_x±s,normal distribution test and homogeneity test of variance were performed before statistics t test was used for comparison between groups before and after statistics,P<0.05 being statistically significant.Results:1.Pictures of rat vaginal secretions after ovariectomy showed that:For five consecutive days,the vaginal secretion smears of rats in the normal group showed the changes in the laws of pre-estru-estrus-post-estrus?interestrus.The other groups of rats showed chaotic irregular changes in the cycle,most of which remained in the interestrous phase.2.HE staining results:HE staining results of superficial fascia:Compared with the model group,there was no significant change in the rats treated with heat-sensitive moxibustion for 4 days,but the change was significant in the rats treated with heat-sensitive moxibustion for 7 days and 14 days.By the action of heat,collagen fibers are pulled and reversible shrinkage of fibroblasts occurs.The connective tissue in the“Mingmen”point area shows that collagen fibers are arranged in a neat and wavy pattern.Changes of collagen fibers after fluorescence staining:Normal group,model group of collagen fiber rows of crisscross the clutter are arranged mesh,and the heat-sensitive moxibustion four days of rat collagen fibers arranged almost no change,as the thermal moxibustion time gradually extended to 14days change significantly,collagen fiber under the effect of heat-sensitive moxibustion stretch traction,tension increases overall assumes the centripetal configuration,namely nearby the“Mingmen”.HE staining results of spinal cord:For the central nervous system,it can promote the proliferation of motor neuron cells and increase the sensitivity of posterior horn cells to heat-sensitive stimulation.By heat-sensitive moxibustion 4 days big rat spinal cord has no obvious change in the model group,the first 7 days heat-sensitive moxibustion anterior cornual neurons number increases and the volume increases,the spinal dorsal horn of glial cell number increase,the heat-sensitive moxibustion anterior horn cells neurons number increased significantly after 14 days,and the volume increase,the nucleus is large and round with obvious nucleoli.3.Sp method observation results:Sp method observation of superficial fascia:SP staining of superficial fastia near“Mingmen”point showed that fibroblasts and macrophages were surrounded by brown and yellow reaction zones.The overall strength and weakness of ERK1/2and P38 proteins in the five groups were as follows:model group<normal group<heat-sensitive moxibustion for 4 days group<heat-sensitive moxibustion for 7 days group<heat-sensitive moxibustion for 14 days group.Comparison of results between“Mingmen”point and“Zusanli”point:After comparison,it was found that the protein expression of heat-sensitive moxibustion“Mingmen”point was higher than that of“Zusanli”point,but did not constitute essential difference(P>0.05).Sp method observation of spinal cord:SP staining showed that the positive reaction of ERK1/2 protein was mostly concentrated around the neurons in the anterior cornual of the spinal cord,while P38protein was more shallow.However,both of them have the most prominent effect under the action of heat-sensitive moxibustion for 14 days,and heat-sensitive moxibustion for 4 days group has no statistical significance(P>0.05).The overall strength and weakness of the model group<heat-sensitive moxibustion for 4 days group<normal group<heat-sensitive moxibustion for 7 days group<heat-sensitive moxibustion for 14 days group.4.Western-blot results:In heat-sensitive moxibustion 4,7,14 days after the“Mingmen”in a nearby muscles of rats ERK1/2 and P38 lightning proteins in general is on the rise,including compared with heat-sensitive moxibustion 4 days group is increased,but no statistical significance(P>0.05),heat-sensitive moxibustion 14 days group compared with normal group and model group increased significantly(P<0.01).5.Serum phosphorus,calcium ion and alkaline phosphatase concentration measurement results:After 14 days of heat-sensitive moxibustion,the concentrations of phosphatase and alkaline phosphatase in rat serum increased(P<0.01),and the concentrations of calcium ions decreased slightly(P<0.05).Conclusion:1.The osteoporosis model can be effectively prepared by OVX to observe the changes in the number of vaginal epithelial cells and white blood cells,so as to prove the success of castration.2.Heat-sensitive moxibustion at“Mingmen”for 14 days is the best time,which can cause loose connective tissue changes in rats with osteoporosis.By the action of heat,collagen fibers are pulled and it can promote the proliferation of motor neuron cells and increase the sensitivity of posterior horn cells to Heat-sensitive stimulation.3.Heat-sensitive moxibustion at“Mingmen”acupoint can improve the expression of ERK1/2 and P38 protein in superficial fascia and spinal cord to ameliorate osteoporosis;in terms of time,the longer the time(14 days),the more obvious the effect.4.Heat-sensitive moxibustion at“Mingmen”Point can improve osteoporosis by increasing ERK1/2 and P38 protein kinase activation of MAPK signaling route in muscle,but heat-sensitive moxibustion for 14 days is the best.5.Heat-sensitive moxibustion can reduce the concentration of calcium ions in the serum,increase the blood phosphorus and alkaline phosphatase,thereby promoting the activity of osteoblasts and inhibiting the proliferation of osteoclasts,which is of great significance for bone reconstruction and healing.In summary,heat-sensitive moxibustion canameliorate the symptoms of osteoporosis by conducting the MAPK signal route through morphological changes of connective tissue and nerve-body fluid regulation.Heat-sensitive moxibustion for 14days is the ideal time.