| BackgroundBotulinum toxin type A(BTX-A)and Hyaluronic acid(HA)gel combined with injection for treating facial rejuvenation is better than HA gel injection alone.At the same time,it was found that BTX-A can significantly reduce the metabolic rate of HA gel in the body and has a significant synergistic effect.However,there is no related literature at home and abroad to report the related mechanism of BTX-A reducing HA gel metabolism.In addition,in clinical operations,the incidence of HA injection complications such as excessive filler dose,filler displacement,vascular embolism,skin tissue necrosis,blindness,etc.is higher.And it is necessary to inject hyaluronidase(HAase)in time to dissolve,but there is no related literature about the effect of BTX-A on the activity of HAase.ObjectivesThis experiment intends to study the effect of BTX-A on the degradation of HA gel in vitro and observe the effect of BTX-A on the metabolism of HA gel in rats,and to explore the mechanism of the synergistic effect of the two combined treatments,and to provide experimental basis for clinical operations.Methods1.Effect of BTX-A on the enzymatic hydrolysis of HA gel in vitroTake 5 parts of Matrifill and Biohyalux HA gel each with a volume of 0.05mL,after staining with 2%toluidine blue for 12 hours and re-warming at room temperature,25?,for 1 hour,add BTX-A 2.5U(2.5 U/mL),5.0U(5.0U/mL),10.0U(10.0U/mL),20.0U(20.0U/mL),and normal saline 1.0mL,respectively.Then add300.0U HAase,and immediately observe the enzymolysis status of the HA gel under a microscope,and record the time to reach the maximum enzymatic hydrolysis state,and collect Images by the microscope imaging system at intervals of 0 to 110 min,and calculate the average optical density(OD)values by ImageJ software.2.Effect of BTX-A on the degradation of HA gel in ratsThe HA filler,Biohyalux,was injected subcutaneously on both sides of the back of the rats with a volume of 0.25 mL.after 5 minutes,BTX-A was injected into the dermis layer of the left at a dose of 5.0U(5.0U/0.1mL)as the BTX-A group,and0.1mL of normal saline was injected on the right as the control group.On the 3rd,7th,14th,30th,60th,and 90th days,the skin tissues and the remaining HA gel were collected from the injection area for detecting the expression of HYAL-1 and HYAL-2in the skin tissues by immunohistochemical staining,detecting the activity of superoxide dismutase(SOD)by xanthine oxidase method,detecting the activity of catalase(CAT)by ultraviolet colorimetry,and detecting the content of malondialdehyde(MDA)by spectrophotometry,and calculating the remaining volume of HA gel by the drainage method,and calculating the metabolic rate of HA gel.Results1.Effect of BTX-A on the enzymatic hydrolysis of HA gel in vitroUnder the action of 0U,2.5U,5.0U,10.0U,20.0U BTX-A,The HA filler of Biohyalux reached the maximum enzymatic hydrolysis state at 60min,70min,80min,100min,and 100min,and the enzymatic hydrolysis rates were 72.663%,68.791%,64.683%,58.599%,58.334%,respectively.The HA filler of Matrifill reached the maximum enzymatic hydrolysis state at 60min,70min,90min,100min,and 100min,and the enzymolysis rates were 63.390%,54.999%,44.965%,41.890%,42.067%,respectively.The dosage of BTX-A is 0-10.0U,with the increase of the dosage,the inhibitory effect on the activity of HAase is gradually increased.The time for the two HA gels to reach the maximum enzymatic hydrolysis state is prolonged,and the enzymatic hydrolysis rate gradually decreases,showing a dose-dependent.When the dose of BTX-A exceeds 10.0U,the inhibitory effect no longer increases.2.Effect of of BTX-A on the degradation of HA gel in ratsDuring the experiment,there was no obvious change in daily activities and living habits of each animal,no abnormal performance such as agitation and burnout,and normal eating and drinking.Before the injection,the skin of the back of each group of rats was intact,and the sides were symmetrical without bulge.After injection of the drugs,the injection area is locally bulged,which has the characteristics of soft touch,clear boundaries,and low mobility,without changes such as erythema and skin ulceration.On the 3rd,7th,and 14th days of the experiment,there was no significant difference in the bulge between the test group and the control group,and the bulge was significantly higher on the 30th,60th,and 90th day than in the control group.On the 3rd day after BTX-A injection,the OD value of HYAL-1 in the skin tissue decreased from 0.256±0.005 to 0.243±0.003 in the control group(t=3.911,P=0.008).on the 7th day,the OD value in the BTX-A group decreased from 0.254±0.006 to 0.240±0.006 in the control group.(t=2.846,P=0.029).after 14 days and later,there was no change in the expression of HYAL-1 in the two groups(P>0.05).On the 3rd day after BTX-A injection,the OD value of HYAL-2 in the skin tissue decreased from 0.264±0.010 to 0.238±0.005 in the control group(t=3.897,P=0.008).on the 7th day,the OD value in the BTX-A group decreased from 0.263±0.003 to 0.236±0.003 in the control group.(t=11.179,P<0.001).On the 14th day,the OD value in the BTX-A group decreased from 0.258±0.006 to 0.233±0.003 in the control group(t=6.165,P=0.001).After 30 days,there was no change in the expression of HYAL-2 in the two groups.(P>0.05).There was no significant difference in the activity of SOD and CAT between the BTX-A group and the control group(P>0.05),and there was no significant difference in the content of MDA between the BTX-A group and the control group(P>0.05).The initial volume of HA gel in the BTX-A group and the control group was447.5±10.9mm~3 and 440.0±14.1mm~3(t=0.728,P=0.496).After 90 days,the remaining volume was reduced to 305.0±11.2mm~3 and 197.5±10.9mm~3(t=11.926,P<0.001),and the metabolic rates were 31.8%and 55.1%,respectively.The metabolic rate of the two groups showed a significant difference after 7 days(P<0.001).Conclusions1.BTX-A has a dose-dependent inhibitory effect on the enzymatic hydrolysis of HA in the dose range of 0-10.0U.2.BTX-A can reduce the expression of HYAL-1(3d,7d)and HYAL-2(3d,7d,14d)in rats and significantly reduce the metabolism of HA gel in vivo.3.Combined injection of BTX-A and HA gel to treat facial rejuvenation,BTX-A was injected first and HA gel was injected 14 days later.When BTX-A is chosen to be injected at the same time and the HAase needs to be dissolved within 14 days after the HA filler injection,the dose of HAase may need to be increased. |
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