| Background:During autologous fat grafting,the harvested fat grafts are typically placed or sedimented in vitro before injection.This period of exposure may affect the viability of adipocytes.To address this issue,a common clinical practice is storing the fat grafts at 4℃or in ice saline.It is believed that there is no cell damage from freeze-thaw cycles and reduced energy consumption of fat cells when stored at 4℃,which is advantageous for short-term storage.On the other hand,cryolipolysis is a technique that takes advantage of the fact that fat cells are more susceptible to cold-induced injury.It targets local fat accumulation areas utilizing a vacuum cooling applicator,inducing inflammation,causing apoptosis or necrosis of fat cells,and ultimately reducing fat cells.In studies related to cryolipolysis,it is generally accepted that the lipolysis effect caused by apoptosis or necrosis can be obtained when the cooling temperature is lower than 10℃.Although fat transplantation and cryolipolysis involve low-temperature techniques,their effects on adipocytes are fundamentally different.This contradiction has not received sufficient attention in research.Furthermore,most studies on cryolipolysis have focused on clinical research,and the mechanisms and influencing factors that trigger fat cell apoptosis in cryolipolysis have yet to be fully elucidated.While significant inflammatory reactions have been observed in clinical studies related to cryolipolysis,the impact and mechanism of inflammation cold-induced injury on adipocytes have been relatively understudied in previous basic research and require further study.Understanding the mechanisms of low temperature on adipocytes is helpful in optimizing the techniques of both fat transplantation and cryolipolysis.It provides a more scientific basis for applying low temperatures in the field of fat.Objectives:1.To explore the effect of in vitro low temperature on the viability of adipocytes and the related mechanism.2.To analyze the mechanism of inflammation in cold-induced injury on adipocytes.3.To conduct experiments in vivo to study the mechanism of low temperature inducing adipocyte apoptosis through a cryolipolysis model of subcutaneous fat in SD rats.Methods:Part Ⅰ:To investigate the effect of low temperature on the viability of adipocytes in vitro.The inguinal fat of rats was obtained and processed into uniformly sized fat particles(n=100),which were divided into five groups:fresh control group(N0),0℃ experimental group(A),4℃ experimental group(B),10℃ experimental group(C),and room temperature(25±1)℃ experimental group(D).After incubating at different temperatures for 2 hours,the fat particles were obtained to evaluate the protein expression of Annexin V and caspase-3.H&E staining.Perilipin-1 immunofluorescence staining,and glucose transfer experiments were performed to assess the effects of different temperatures on the viability of adipose tissue in vitro.Part Ⅱ:To investigate the effect of low temperature on the immune homeostasis of adipose tissue in vitro.The inguinal fat of rats was obtained and processed into uniformly sized fat particles(n=80),which were divided into five groups:fresh control group(N0).0℃experimental group(A).4℃ experimental group(B),10℃ experimental group(C),and room temperature(25±1)℃ experimental group(D).After incubation at different temperatures for 2 hours,the fat particles were obtained to observe the effect of low temperature on the homeostasis of immune function in adipose tissue through suspension array and real-time fluorescent quantitative PCR.Part Ⅲ:To establish a cryolipolysis model of subcutaneous fat in rats and to explore the mechanism of local low temperature on adipocytes in vivo.A stainless steel ice block was applied to the inguinal region on one side of the rat(n=40),and the contralateral side was only exposed as the control side.The treated ice blocks were changed every 5 minutes to maintain the ice temperature at 0℃.lasting 1 hour.On days 0(1 hour post-treatment),3.7,and 30 after low-temperature stimulation,inguinal adipose and skin tissues from the treated and untreated side regions(n=8)were obtained to evaluate the protein expression of MCP-1,IL-6,IL-1β,and PPAR-δ.H&E staining and Perilipin-1 immunofluorescence staining were performed to assess the effects of low-temperature stimulation on the viability,inflammation,and lipid metabolism pathway in adipose tissue.Results:Part Ⅰ:Isolated adipose tissues were incubated in different temperature environments,and the rate of adipocyte membrane disruption at 4℃ was significantly higher in the 0℃ and room temperature groups.The viable adipocytes were significantly lower in the 4℃group than in the room temperature group.The isolated adipose tissues all showed a significant decrease in glucose transfer.Compared with the fresh tissue,the decrease in glucose transfer was most evident in the 4℃ group,followed by 10℃,0℃,and room temperature groups.The expression of Annexin V and Caspase-3 showed an elevated trend in all groups,while the expression was significantly higher in the 4℃ group.Part Ⅱ:Compared with the low temperature of 0℃,4℃,and 10℃ groups,the room temperature group had higher expression of pro-inflammatory adipokines of MCP-1 and IL-6.The expression of TNF-α and IL-1β was lower and did not change significantly in all groups.The mRNA expression level of MCP-1 in adipose tissue was significantly higher in the room-temperature group than in the low-temperature group,and the mRNA expression levels of other inflammatory substances IL-6,IL-1β,and TNF-α did not differ significantly among the groups.Part Ⅲ:The infiltration of Immune cells on the treated side began to appear as early as Day 0.gradually increasing between Days 3 and 7,and resolving on Day 30 after cold treatment.No significant immune cell infiltration was observed on the untreated side.After cold treatment,different degrees of adipocyte damage was seen in certain areas on Day 0,Days 1,3,and 7,and adipocytes were restored to normal on Day 30.Inflammatory substances MCP-1.IL-6.and IL-1β on the treated side started to increase as early as Day 0 and then gradually decreased and resolved on Day 30 after cold treatment.At the same time,the untreated side also showed an increase in inflammatory substance.but the response lagged behind the treated side and with a generally mild degree.The expression of PPARδ on the treated side showed an increasing trend at Days 0,1,and 3 after cold treatment,followed by a decreasing trend at Days 7 and 30,and the expression on Day 1 was significantly higher than that of the untreated side.The overall protein expression level of the treated side was higher than that of the untreated side.Conclusions:Isolated fat particles all showed varying degrees of decreased viability in vitro.However,the low temperature of 0℃、4℃、10℃ caused the most significant cold injury of adipose tissue.The damage effect of low temperatures above 4℃ on adipocytes is mainly achieved by inducing adipocyte apoptosis,and 4℃ group showed the highest level of apoptosis.Adipose tissue exhibited increased expression of pro-inflammatory adipokines at room temperature in vitro.At the same time,the homeostasis of immune function did not differ in adipose tissue under low temperatures of 0℃,4℃,and 10℃ compared with fresh adipose tissue.In the rat model,the inflammatory response was more obvious in the early phase(Days 0,3 after treatment),started to decline on Day7 and resolved in the late phase(Day 30),while regeneration from ADSCs present in adipose tissue started to occur on Day 30.Meanwhile,the low-temperature stimulation may activate endogenous lipid metabolism through the PPARδ pathway,thus affecting lipid catabolism. |
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