| Traditional Chinese medicine has a long history and rich experience in the treatment of pain of phlegm and blood stasis,and there are differences in the treatment of different sputum pains.In this experiment,the rat model of phlegm and blood stasis caused by complete Freund’s adjuvant was used.At the same time,the cold and damp heat factors were introduced on the basis of the phlegm syndrome model,and the traditional Chinese medicine Kuanjin vine intervention was used to analyze the therapeutic effect of traditional Chinese medicine on different phlegm and blood stasis syndrome.The evaluation of the analgesic mechanism at the central sensitization level reflects the significance of TCM syndrome differentiation.1.Study on anti-inflammatory and analgesic effects of broad-necked vineObjective:To explore the mechanism of analgesic and anti-inflammatory effects of Tinospora sinensis,and to analyze its mechanism of actionMethods:After intradermal injection of complete Freund’s adjuvant in the foot and ankle,they were divided into 7 groups according to random number method,normal group,model group,prednisone group,Tinospora sinensis water extract 1.2.g/kg,2.4g/kg group,Tinospora sinensis alcohol extract 1.2.g/kg,2.4g/kg group.Each group began the first intragastric administration 24 hours after model establishment,once a day,for a total of 12 days.After 2 hours of the first administration,the body weight,the 50%mechanical contraction pain reaction value,the heat-shrinking reaction time,and the swelling rate of the foot and ankle were measured,and then measured every other day.After 12 days of administration,the materials were perfused and immunohistochemical sections of the spinal cord were made to observe the expression of NMD A receptors.Serum IL-1α、IL-6,IL-10 and TNF-a were detected by enzyme-linked immunosorbent assay(ELISA).Results:①Paw swelling rate:the swelling rate of model animals increased significantly after modeling(compared with the control group,P<0.01),and the degree of swelling reached the peak on the 3rd day after modeling.The doses of aqueous extracts and alcohol extracts of the Tinospora sinensis significantly reduced the degree of swelling of the ankles of rats in the first and third days after modeling(compared with the model group,P<0.01),and the water extract of Tinospora sinensis 1.2 g/kg,alcohol extract 2.4g/kg was significantly lower than the model group at the other time points(compared with the model group,P<0.05,P<0.01).②Heat-shrinking reaction time:After the model was established,the reaction time of the model animals was significantly shortened(compared with the control group,P<0.01),and reached the lowest value on the third day after modeling.The alcohol extract of Tinospora sinensis 1.2g,2.4g/kg and the water extract of Tinospora sinensis 1.2g,2.4g/kg can significantly prolong the pain reaction time of rats within 12 days after model establishment(compared with the model group,P<0.01).③50%mechanical shrinkage reaction value:50%mechanical contraction of the model animal was significantly reduced after modeling(compared with the control group,P<0.01),and reached the lowest point on the third day after modeling.The doses of the aqueous extracts and alcohol extracts of the Tinospora sinensis significantly increased the 50%mechanical shrinkage response of the animals after 1 hour of administration(compared with the model group,P<0.01),and the rats were significantly increased within 12 days after modeling.50%mechanical contraction response(compared to the model group,P<0.01).④Inflammatory cytokines:serum inflammatory cytokines IL-1α,TNF-α,IL-6 levels in model rats were significantly increased(compared with normal group,P<0.05,P<0.01),and IL-10 levels were significantly decreased(compared with normal group,P<0.05,P<0.01).After 12 days of administration,the doses of the aqueous extracts and alcohol extracts of the Tinospora sinensis could significantly reduce serum IL-1α,TNFα and IL-6 levels in model rats.The IL-10 content in the model rats was increased(compared with the model group,P<0.05,P<0.01),which had a certain therapeutic effect on inflammatory pain.⑤NMDA receptor expression in the spinal cord:After modeling,the expression of NMDA receptor in the spinal dorsal horn of the injured side of the inflammatory pain model rats was significantly increased(compared with the normal group,P<0.01).The doses of the aqueous extracts and alcohol extracts of the Tinospora sinensis can significantly reduce the expression of NMDA receptor in the spinal cord of rats with inflammatory pain model(compared with model group,P<0.01).Conclusion:Both the water extract and the alcohol extract of the Tinospora sinensis can effectively inhibit the sensitivity of the inflammatory pain model rats to temperature and mechanical stimuli;the analgesic and anti-inflammatory mechanism of the Tinospora sinensis reduces the peripheral inflammatory factors and inhibits the spinal cord The expression of the horn NMDA receptor is related;the analgesic and anti-inflammatory effects of the aqueous extract and the alcohol extract of the Tinospora sinensis are basically the same,so the subsequent experiment uses the Tinospora sinensis water extract.2 Study on the effect of 2 gluten vine on the pain sensitivity of cold and hot sputum ratsObjective:To observe the intervention effect and effect of Tinospora sinensis on hyperalgesia and phlegm-sensing model rats,and analyze its action principle from the perspective of central sensitization of pain sensation.Methods:According to the random number method,it was divided.into 11 groups,normal group,model group,positive drug group,hot pain model group,Tinospora sinensis enthalpy model 0.6g/kg,1.2g/kg,2.4g/kg group,Tinospora sinensis cold mode10.6g/kg,1.2g/kg,2.4g/kg group.After intradermal injection of complete Freund’s adjuvant in the foot and ankle,the model of the cold and heat sputum and the drug-administered group were placed in a constant temperature climate chamber for 4 hours after the inflammatory model was established.Each administration group began the first intragastric administration for 14 days 24 hours after the plantar model.Before the modeling and 3d,7d,10d and 14d after modeling,50%mechanical pain reaction value and heat shrinking reaction time index were measured.Immunohistochemical sections of the spinal cord and the dorsal root ganglia were made on the day after the last administration,and the expression of CD11b,CX3CL1,CX3CR1 and NMDA receptors were observed.Rats were harvested after the last dose,and the remaining six fresh silk penins in each group were placed in a cryotube for rapid freezing.The expression of CX3CL1,CX3CR1 and NMDA receptors in rat spinal cord was determined by Western blot.Results:①Heat-shrinking reaction time;The pa:in response time of the model rats was significantly lower(compared with the baseline values of the same group,P<0.01),suggesting that the sensitivity of the rats’ foot to temperature stimulation was significantly enhanced after modeling.The pain reaction time of the model group rats was significantly lower than that of the control group at each time point after the model establishment(compared with the control group,P<0.01),and reached the lowest on the 7th day after modeling,but it has no difference on the 14th day after modeling.See obvious recovery.The participation of cold factors can inhibit the sensitivity of rat foot to temperature stimulation to varying degrees,and the effect on the 3rd,7th and 10th day after modeling is significant(compared with the model group,P<0.05,P<0.01).The participation of damp heat factor can prolong the pain response time of model rats on the third day(compared with the model group,P<0.05),and the other time points are not obviorus;the rats in the cold sputum model group have the pain response time after modeling 7th,10th and 14th were significantly longer than those of the sputum model rats(compared with the sputum model group,P<0.05,P<0.01).The various dose groups of Tinospora sinensis can prolong the pain response time of the rats in the enthalpy model.The 2.4g/kg of the Tinospora sinensis is 1.2 g/kg on the 14th day after the administration.On 7th,10th and 14th the gluten-free vine 0.6g/kg was effective on the 7th day after administration(compared with the sputum model group sensitivity of chilling model rats to temperature stimulation,P<0.05,P<0.01).②50%mechanical contraction response:50%mechanical pain response in model rats was significantly reduced(compared with the baseline values of the same group,P<0.01).The 50%mechanical pain response of the rats in the model group at each time point after modeling was significantly lower than that of the control group(compared with the control group,P<0.01),and 50%mechanical pain response value on the 7th day after modeling.At the lowest level,no significant recovery was seen on the 14th day after modeling.On the third day after the introduction of cold and damp heat factors,the sensitivity of the rats’ foot to mechanical stimulation was significantly reduced(compared with the model,P<0.05,P<0.01).On the 7th and 10th day,the rats suffered from the foot.The sensitivity to mechanical stimuli did not change much,but there was a significant difference between the chilling model group and the enthalpy model group(between the enthalpy model group and the chilling model group,P<0.05).On the 14th day after modeling,the introduction of cold and damp heat factors can reduce the 50%mechanical pain response of rats to different degrees compared with the model group.The participation of cold and dampness factors makes the rats’ foot more mechanically stimulated.For sensitivity,the performance was reduced by 50%mechanical pain response(compared to the model group,P<0.05).The different doses of the gluten vine can increase the 50%mechanical pain response value of the sputum model rats in different degrees.Among them,2.4g/kg of gluten vines on the 7th,10th and 14th day after administration,1.2g/kg of gluten free vines On the 10th and 14th day after administration,the effect was obvious(compared with the sputum model group,P<0.01);the different doses of the Tinospora sinensis could increase the sensitivity of the cold sputum model rats to mechanical stimulation,showing 50%.The MWT values were reduced to different extents.Among them,2.4g/kg of Tinospora sinensis at different time points after administration,1.2g/kg of Tinospora sinensis had obvious effect on the 14th day after administration(compared with the cold sputum model group P<0.05,P<0.01).There were also different degrees of difference in the same dose of the glutinous vine tempering model group and the chilling model group.Among them,2.4g/kg of Tinospora sinensis was applied at each time point after administration,and 1.2g/kg of Tinospora sinensis was administered.After the 7,h,10th and 14th days,the difference was obvious(compared with the same dose of the gluten-free glutinous tempering model group and the chilling model group P<0.05,P<0.01).③Western blotting method to determine the expression of receptors in the spinal cord:The expression of CX3CR11 CX3CL1 and NMD A receptor proteins in the spinal cord of the model rats was significantly increased(compared with the control group,P<0.01).The cold factors and the participation of damp heat factors can significantly increase the expression of CX3CR1,CX3CL1 and NMDA receptor proteins in the spinal cord of model rats(compared with the model group,P<0.05),and the effect of damp heat factor is more obvious.The expression of CX3CR1,CX3CL1,and NMDA receptor proteins in the spinal cord of each model group and zephyr model group was significantly reduced after sinensis aqueous extract(compared with the model group,P<0.01).④Immunohistochemical method for the determination of receptor expression in the spinal cord:The expression of CD11b,CX3CR1,CX3CL1 and NMDA receptor proteins in the spinal cord of the model rats was significantly increased(compared with the control group,P<0.01).The cold factors and the participation of damp heat factors can increase the expression of CD11b,CX3CR1,CX3CL1 and NMDA receptor proteins in the spinal cord of model rats,and the effect of cold factors is more obvious.The expressions of CX3CR1,CX3CL1 and NMDA receptor proteins in the spinal cord of the model group and the zephyr model group were significantly decreased after(compared with each dose of the aqueous extract of the glutinous rice vines,P<0.05,P<0.01).⑤Immunohistochemical method to determine the expression of receptors in the dorsal root ganglia;The expression of CD11b,CX3CR1,CX3CL1,NMDA receptor protein in the rat dorsal root ganglia was significantly increased(compared with the control group,P<0.01).The cold factors and the introduction of damp heat factors can increase the expression of CD11b,CX3CR1,CX3CL1 and NMDA receptor proteins in the dorsal root ganglia of the model rats,and the effect of cold factors is more obvious.The expressions of CD11b,CX3CR1,CX3CL1 and NMDA receptor proteins in the dorsal root ganglia of rats in each enthalpy model group were significantly reduced after each dose of the aqueous extract of the glutinous vines group(compared with the model group,P<0.05,P<0.01).Conclusion:The aqueous extract of sinensis can effectively reduce the high sensitivity of temperature and mechanical stimulation in rats with sputum,and its mechanism is related to the pathway of CD11b,CX3CR1,CX3CL1 and NMDAR in the spinal cord of rats with sputum syndrome.There may be synergistic effects between several chemokines,attenuating the effect of a chemokine and simultaneously attenuating the expression of various chemokines.In addition、the same dose of Tinospora sinensis water extract is cold and hot.The treatment effect of phlegm and blood stasis syndrome is not the same.On the whole,the cold-cold drug Tinospora sinensis has better therapeutic effect on enthusiasm,and the theoretical basis of syndrome differentiation and treatment of Chinese medicine has been confirmed again. |
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