| Objective:We dynamically observed changes in toll-like receptor 2 and 4(TLR2 and TLR4),high mobility group box 1(HMGB1)and Interleukin-10(IL-10)expression after catgut and poly-glycolide-co-lactide(PGLA)thread embedding in the Zusanli acupoint area of rats.To explore the mechanism underlying acupoint thread embedding stimulation as well as the timeline of the effect,and to lay the foundation for further exploring the initial response mechanism of acupoint thread embedding.Methods:A total of 160 male Sprague–Dawley rats were randomly divided into a “blank”group(n=10),an “embedding needle empty-prick” group(ENE,n=50),a “catgut embedding” group(CE,n=50),and a “PGLA thread embedding” group(PTE,n=50).The three intervention groups were randomly subdivided into five time point groups,8h,3 d,7 d,14 d,and 21 d after intervention,with 10 animals in each group.Rats were embedded once with 2-0*5-mm catgut or PGLA threads or pricked once with a buried needle without a thread.The skin and subcutaneous connective tissue of the acupoint area were collected at the corresponding time points.TLR2,TLR4 and IL-10 levels were assessed with enzyme-linked immunosorbent assay and HMGB1 expression with western blotting.Results:1.General Conditions.The rats in each group exhibited good mental status and mobility.No redness,swelling,pus,or ulceration was observed in the Zusanli area of any rat.2.TLR2 Contents.(1)Compared with the blank group,TLR2 levels were significantly increased in the CE or PTE group at each time point(P<0.001,P<0.01).The increase began at 8 h after embedding,reached its peak at 3 d in the CE group and at 7 d in the PTE group,then gradually decreased.At 21 d,the two embedding groups still showed higher levels than the blank group(P<0.05),TLR2 levels at 3 d,14 d,and21 d in the CE group and in the PTE group at 14 d significantly differed from those at the respective previous time point(P<0.001);TLR2 levels were significantly increased in the ENA group at 8 h,3 d,and 7 d(P<0.001,P<0.05),TLR2 levels in the ENA group decreased gradually after reaching a peak at 8 h and decreased to the level of the blank group at 14 d and 21 d(P>0.05).At 3 d and 7 d in the ENA group significantly differed from those at the respective previous time point(P<0.01,P<0.05).(2)TLR2 levels in the PTE group were lower than those in the CE group at the same time point,all differences were statistically significant(P<0.05).3.TLR4 Contents.(1)Compared with the blank group,the content of TLR4 significantly increased in the CE or PTE group at each time point(P<0.001,P<0.01).Levels began to increase at 8 h after embedding,reached a peak at 3 d in the CE group and at 7 d in the PTE group,and then gradually decreased.At 21 d,levels were still higher in the two embedding groups than in the blank group(P<0.05).TLR4 levels at 3d,7 d,14 d,and 21 d in the CE group,and at 7 d,14 d,and 21 d in the PTE group significantly differed from the respective previous time point(P<0.001,P<0.01);the content of TLR4 significantly increased in the ENA group at 8 h,3 d,7 d,and 14 d(P<0.001,P<0.05).Contents of TLR4 in the ENA group decreased to the levels of the blank group at 21 d(P>0.05).TLR4 levels at 7 d,14 d,and 21 d in the ENA group significantly differed from the respective previous time point(P<0.01,P<0.05).(2)TLR4 levels in the PTE group were lower than those in the CE group at the same time point,all differences were statistically significant(P<0.05).4.HMGB1 Expression.(1)Compared with the blank group,HMGB1 levels significantly increased in the CE or PTE group at 8 h,3 d,7 d,and 14 d(P<0.001,P<0.01,P<0.05).They began to increase at 8 h after embedding,reached a peak at 3 d in the CE group and at 7 d in the PTE group,and then gradually decreased.At 21 d,levels in the two embedding groups had decreased to the level of the blank group(P>0.05).HMGB1 levels at 3 d,7 d,and 14 d in the CE group,14 d and 21 d in the PTE group significantly differed from the respective previous time point(P<0.001,P<0.01,P<0.05);HMGB1 levels significantly increased in the ENA groups at 8 h,3 d,7 d,and14 d(P<0.01,P<0.001),there was no significant difference at 21 d(P>0.05).HMGB1 levels at 7 d in the ENA group significantly differed from the level at 3 d(P<0.05).(2)HMGB1 levels in the PTE group were lower than those in the CE group at the same time point,except for the 7 d time point,when levels were slightly higher in the PTE group.HMGB1 levels at 3 d differed significantly between the CE and the PTE group(P<0.05).5.There was an extremely strong positive correlation between TLR2,TLR4 and HMGB1 in all intervention groups(all r > 0.8,P < 0.01).6.IL-10 Contents.(1)Compared with the blank group,the content of IL-10 significantly increased in the CE group at each time point and in the PTE group at 8 h,3d,7 d,and 14 d(P<0.001,P<0.01).Levels began to increase at 8 h after embedding,the CE group reached the peak at 3d and it was still higher than the blank group at 21d(P<0.001),the PTE group reached the peak at 7d and then decreased to the level of the blank group(P>0.05).IL-10 levels at 3 d,7 d,14 d,and 21 d in the CE group,14 d and21 d in the PTE group significantly differed from the respective previous time point(P<0.001,P<0.05);The content of IL-10 significantly increased in the ENA group at 8h,3 d,and 7 d(P<0.001),and decreased to the levels of the blank group at 14 d and 21d(P>0.05).IL-10 levels at 3 d and 7 d in the ENA group significantly differed from the respective previous time point(P<0.001,P<0.05).(2)IL-10 levels in the PTE group were lower than those in the CE group at the same time point,all differences were statistically significant(P<0.05).Conclusions:Thread type is the main factor causing the parabolic dynamic changes in microenvironment cell chemistry of the acupoint area,and there are some differences between the threads.The activation of TLR2-and TLR4-mediated inflammatory signaling pathways by HMGB1,and the anti-inflammatory factor IL-10 may be involved in the local stimulation effect observed at acupoints after embedding,and the moderate inflammation dynamic changes may be an important part of the source initiation mechanism underlying the acupoint thread embedding effect. |
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