| Objective: Various inflammatory cytokines play a central role in the occurrence and development of SIRS,and Gua Sha therapy has a regulatory role on inflammatory cytokines.This study dynamically analyzed the inflammatory process from the time dimension,and explored the effect of Gua Sha with different doses of frequencies at different times on the survival rate and related inflammatory cytokines of SIRS rats.Methods: In experiment 1,SPF healthy adult Wistar rats weighing 200 ± 10 g after fasting for12 h were divided into blank group,400mg/kg ZY group,500mg/kg ZY group,600mg/kg ZY group and 700mg/kg ZY group according to the digital random table method,with 16 rats in each group,a total of 80 rats.The blank group was given intraperitoneal injection of sterile paraffin oil to rats.The 400mg/kg ZY group,500mg/kg ZY group,600mg/kg ZY group and700mg/kg ZY group were given intraperitoneal injection of 400mg/kg,500mg/kg,600mg/kg and 700mg/kg zymosan suspension to rats,respectively.Six rats were randomly selected from each group to measure rectal temperature before and 6 and 24 hours after modeling,and the peripheral blood leukocyte count was measured by automatic blood analyzer 24 hours after modeling,The lung,liver,spleen and kidney were taken for HE pathological examination,and the survival rate was not included in the observation.The survival rate of the remaining 10 rats in each group was observed and recorded at 6,24,48,72 and 96 hours after modeling.In experiment 2,SPF healthy adult Wistar rats weighing 200 ± 10 g after fasting for 12 h were divided into model group and scraping group according to digital random table method,with54 rats in each group,a total of 108 rats.(1)The model group was randomly divided into 9groups: Pre-ZY,ZY-1h,ZY-2h,ZY-4h,ZY-6h,ZY-8h,ZY-12 h,ZY-16 h and ZY-24 h groups,6 in each group,54 in total;All rats in the model group were modeled according to experiment1(no intraperitoneal injection of zymosan suspension in Pre-ZY group),and no other intervention measures were involved.The rats in each group took blood from the orbital venous plexus at the corresponding time points(1,2,4,6,8,12,16 and 24 hours before and after modeling),and detected serum TNF-α by enzyme-linked immunosorbent assay(ELISA)、IL-6 content.(2)The Gua Sha group was randomly divided into 9 groups: Pre-GS,GS-1h,GS-2h,GS-4h,GS-6h,GS-8h,GS-12 h,GS-16 h,GS-24 h groups,6 in each group,54 in total;The objects of the Gua Sha group were healthy rats,and they were not given intraperitoneal injection of yeast polysaccharide suspension,but only Gua Sha intervention(no Gua Sha in the Pre-GS group).The rats in each group took blood from the orbital venous plexus at the corresponding time points(1,2,4,6,8,12,16 and 24 hours before and after the Gua Sha)and detected the serum IL-6 content by ELISA.Through depicting the changes of inflammation and Gua Sha through the trend of key inflammatory cytokines within 24 hours,we can get the "aggravation point of inflammation","peak point of inflammation" and "peak point of Gua Sha effect",and draw up the "characteristic timing" of Gua Sha intervention.In experiment 3,SPF healthy adult Wistar rats weighing 200 ± 10 g after fasting for 12 h were divided into blank group,model group,Gua Sha 1 time group,Gua Sha 2 times group and Gua Sha 3 times group according to the digital random table method.There were 16 rats in each group,a total of 80 rats.All groups were modeled according to experiment 1.The blank group and the model group only simulated the Gua Sha,and the Gua Sha 1 time group,the Gua Sha2 times group,and the Gua Sha 3 times group started the Gua Sha at the "characteristic time" according to the results of experiment 2.The Gua Sha 2 times group performed the second Gua Sha at an interval of 1 hour after the first Gua Sha;The Gua Sha 3 times group performed the second Gua Sha at an interval of 1 hour after the first Gua Sha,and the third Gua Sha at an interval of 1 hour.6 rats in each group were randomly selected at the "point of aggravation of inflammation".Blood was collected from the orbital venous plexus and serum was separated.The key cytokine TNF-α、IL-6 content was detected by ELISA;Take samples from the "peak point of inflammation" to detect serum TNF-α 、 IL-6 、 IL-1β 、 IL-10 and viscera-related inflammatory cytokine IL-6,and the histological HE pathological analysis of each organ were not included in the survival rate observation.The survival rate of the remaining 10 rats in each group was observed and recorded at 6,24,48,72 and 96 hours after modeling.In experiment 4,SPF healthy adult Wistar rats weighing 200 ± 10 g after fasting for 12 h were divided into blank group,model group and immediate Gua Sha group according to digital random table method,with 16 rats in each group,a total of 48 rats.The model making method of each group is referred to experiment 1,and the Gua Sha times of the immediate Gua Sha group are referred to experiment 4.According to the results of experiment 2,6 rats in each group were randomly selected to collect blood from abdominal aorta at the "peak point of inflammation",and the serum was separated to detect the relevant inflammatory cytokines TNF-α、IL-6、IL-1β、IL-10 by ELIS was not included in survival observation.The survival rate of the remaining 10 rats in each group was observed and recorded at 6,24,48,72 and 96 hours after modeling.Results: 1.The survival rate of each group in experiment 1: compared with the blank group,400mg/kg ZY group and 500mg/kg ZY group,the survival rate of 600mg/kg ZY group and700mg/kg ZY group was significantly reduced(P<0.01),but there was no significant difference between the survival rate of 600mg/kg ZY group and 700mg/kg ZY group(P>0.05).2.Results of rectal temperature measurement: The rectal temperature of the 600 mg/kg ZY group and 700 mg/kg ZY group was significantly lower than that of the 400 mg/kg ZY group(P<0.01),and also significantly lower than that of the control group(P<0.05).Compared with the blank group,the rectal temperature of the 400 mg/kg ZY group was significantly higher 24 hours after modeling(P<0.01);Compared with 400mg/kg ZY group,rectal temperature in500mg/kg ZY group,600mg/kg ZY group and 700mg/kg ZY group decreased significantly(P<0.05).3.Results of peripheral blood leukocyte count: 24 hours after modeling,compared with the blank group,the peripheral blood leukocyte count of 500 mg/kg ZY group,600 mg/kg ZY group and 700 mg/kg ZY group were significantly lower(P<0.05,P<0.01).4.Histopathological observation results of lung,liver,spleen and kidney: 24 hours after modeling,the lung tissue structure of rats was disordered,the alveolar wall was thickened,the interstitial congestion and edema of the lung,and the interstitial inflammatory cell infiltration of the lung;The liver cells are swollen,degenerated,disorderly arranged,the blood vessel walls of the central vein,hepatic sinus and portal vein are congested,and the liver tissues are covered with inflammatory cells;The spleen tissue structure is unclear,the boundary between red pulp and white pulp is unclear,and macrophages are densely distributed;There are obvious glomerular atrophy,inflammatory cell infiltration and some renal tubulointerstitial edema.However,with the increase of the dosage of yeast polysaccharide,the injury degree of lung,liver,spleen and kidney tissue of rats gradually increased,and the injury was most obvious in the 600 mg/kg ZY group and 700 mg/kg ZY group.5.In experiment 2,the content of serum TNF – α of rats within 24 h after modeling was the highest at 2h after modeling,and significantly increased compared with that before and 1h,4h,6h,and 24 h after modeling(P<0.01);The content of serum IL-6 was the highest at 12 h after modeling,and significantly increased compared with that before modeling and 1h,2h,4h,6h,8h,16 h,and 24 h after modeling(P<0.01).In addition,within 24 hours after Gua Sha,the content of serum IL-6 in rats was the highest at 8 hours after Gua Sha,which was significantly higher than that before Gua Sha and 1h,2h,4h,12 h,16h,and 24 h after Gua Sha(P<0.01).It is suggested that the intervention of Gua Sha 6 hours before modeling can make the "maximum point of Gua Sha effect" fall on the "point of aggravation of inflammation".6.The survival rate of each group in experiment 3: compared with the blank group,the survival rate of the Gua Sha 1 time group,the Gua Sha 2 times group and the model group were significantly lower(P<0.01);Compared with the model group,the survival rate of the Gua Sha group was significantly improved(P<0.01).7.Test results of serum inflammatory cytokines:(1)Compared with the model group,the serum TNF-α in the Gua Sha 2 times group and the Gua Sha 3 times group was higher than that in the model group The level decreased significantly(P<0.05);The serum IL-6 level in the three Gua Sha groups was significantly lower than that in the model group and the first Gua Sha group(P<0.05).At the "peak point of inflammation",compared with the model group,serum IL-6 expression was significantly reduced in the Gua Sha 1 time group,Gua Sha 2 times group and Gua Sha 3 times group(P<0.01);serum IL-1β expression was significantly reduced in the Gua Sha 3 times group compared with the model group(P<0.05),and serum antiinflammatory factor IL-10 expression was significantly increased in the Gua Sha 3 times group compared with the model group(P<0.01).The expression of serum IL-10 was significantly lower in the three Gua Sha groups compared with the model group(P < 0.05).Compared with the group scraped once,the group scraped three times significantly reduced the expression of serum IL-6 and IL-1β(P < 0.01)and increased the expression of serum anti-inflammatory factor IL-10(P < 0.01).Compared with the Gua Sha 2 times group,the Gua Sha 3 times group could significantly reduce the expression of serum IL-6(P < 0.05)and increase the expression of serum anti-inflammatory factor IL-10(P < 0.01)in rats.8.Immunohistochemical results: at "peak inflammation point"(1)Rat lung tissue,compared with the model group,Gua Sha 1 time group,Gua Sha 2 times group,Gua Sha 3 times group IL-6protein positive mean optical density were significantly reduced(P<0.01);compared with Gua Sha 1 time group,Gua Sha 2 times group,Gua Sha 3 times group IL-6 protein positive mean The mean optical density of IL-6 protein positivity was significantly reduced in both the Gua Sha 2 and Gua Sha 3 groups compared with the Gua Sha 1 group(P<0.01);and the mean optical density of IL-6 protein positivity was significantly reduced in the Gua Sha 3 group compared with the Gua Sha 2 group(P<0.01).((2)Rat liver tissue,compared with the model group,the mean optical density of IL-6 protein positive was significantly reduced in the Gua Sha 2 times group and the Gua Sha 3 times group(P<0.01);while the mean optical density of IL-6 protein positive was significantly reduced in the Gua Sha 3 times group compared with the Gua Sha 1times group(P<0.01).(3)Rat spleen tissue,compared with the model group,the mean optical density of IL-6 protein positivity was significantly reduced in the Gua Sha 1 time group,the Gua Sha 2 times group and the Gua Sha 3 times group(P<0.01);in addition,the mean optical density of IL-6 protein positivity was significantly reduced in the Gua Sha 3 times group compared with the Gua Sha 1 time group and the Gua Sha 2 times group(P<0.01).(4)Rat kidney tissues,compared with the model group,only the mean optical density of IL-6 protein positivity was significantly reduced in the Gua Sha 3 times group(P<0.01);in addition,the mean optical density of IL-6 protein positivity was significantly reduced in the Gua Sha 3 times group compared with the Gua Sha 1 times group(P<0.05)9.Histopathological observation results of lung,liver,spleen and kidney: at the "peak point of inflammation",the lung,liver,spleen and kidney tissues of rats in the model group showed the same performance as that in experiment 1.With the increase of Gua Sha times in each Gua Sha group,the degree of inflammatory damage in lung,liver,spleen and kidney of rats was gradually reduced compared with that in the model group,and the improvement was most obvious in the three Gua Sha groups.10.The survival rate of each group in experiment 4: compared with the blank group,the survival rate of the model group and the immediate Gua Sha group was significantly lower(P<0.01,P<0.05);Compared with the model group,the survival rate of the immediate Gua Sha group had an upward trend,but there was no statistical difference(P>0.05).11.Test results of serum inflammatory cytokines: compared with the model group,serum IL-1β in the immediate Gua Sha group was significantly decreased(P<0.01).The serum proinflammatory cytokines TNF-α 、 IL-6 of rats in the immediate Gua Sha group showed a downward trend compared with the model group,and the serum anti-inflammatory cytokine IL-10 showed an upward trend compared with the model group,but there wer no statistical difference(P>0.05).Conclusions: 1.The rat SIRS model was induced by intraperitoneal injection of suspension in a dose-dependent manner.2.Intervention of Gua Sha 6 hours before modeling can make the "maximum effect point of Gua Sha" fall on the "point of inflammation intensification",and at this time,the amount of stimulation of three times of Gua Sha can significantly improve the survival rate of SIRS model rats and reduce the serum inflammatory cytokines IL-6、IL-1β and TNF-α than one or two times of Gua Sha,increase the expression of serum anti-inflammatory cytokine IL-10,and more effectively reduce the inflammatory damage of various organs and tissues.3.The immediate intervention of three times of Gua Sha after model establishment can not significantly improve the survival rate of SIRS model rats,nor significantly reduce the serum proinflammatory factors IL-6 and TNF-α,and increase the expression of serum antiinflammatory cytokine IL-10.4.Different times of interventional Gua Sha have various results on SIRS model rats.The Gua Sha therapy with a certain amount of frequency before modeling has a relatively better intervention effect than the Gua Sha therapy immediately after modeling. |
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