| Liver is one of the most vigorous organs in metabolism of substance and energy,so it has a high sensitivity to ischemia and hypoxia.Therefore,during liver surgery,I/R injury caused by blocking and restoring blood flow is an important factor causing liver injury.Related studies in recent years have shown that ERS plays an important role in liver I/R injury.Therefore,intervention of ERS response levels may become a new method for treating liver I/R.ADSC-CM is derived from ADSCs and contains many types of active substances,which can exert protective effects in various injuries.In recent years,it has been found that ADSCs can reduce liver I/R damage by inhibiting oxidative stress,apoptosis and ERS.However,no research has been reported on the effects of ADSC-CM on ERS and Lipid anabolic during liver I/R combined partial resection.In this study,a laparoscopic minimally invasive technique was used to establish a miniature pig liver I/R combined partial resection injury model,and treated with ADSC-CM.The effects of ADSC-CM on ERS and Lipid anabolic in this model were investigated by observing the ultrastructure of hepatocytes and detecting changes in ERS and Lipid anabolic-related indicators.In this study,twenty-four healthy miniature pigs aged from 4 to 6 months(20 to 30 kg)were randomly divided into IRI group,DMEM group,CM group and ADSCs group,with 6 groups in each group.All groups underwent laparoscopic right hepatic ischemia for 60 min and left hepatectomy.Immediately after surgery,the four groups were injected with the same amount of normal saline,DMEM medium,ADSC-CM and ADSCs.Serum samples and liver tissue were collected before operation,1 day,3 days and 7 days after operation.Observe the changes of ultrastructure of liver cells and oil red O staining.Detection of changes in serum lipid anabolic indicators(TG,CHOL,HDL-C,LDL-C).The changes of lipid anabolic indexes(TG,CHOL,HDL,VLDL,ACC1)in liver were detected by ELISA.The levels of ERS-related genes(GRP78,ATF6,IRE1,PERK,ATF4,CHOP)and lipid anabolic-related genes(SREBP1,SCAP,LXR,FASN,ACC1,HMGCR,HMGCS1,HMGCS2)in liver tissues were detected by q RT-PCR.Western Blot method was used to detect the changes of ERS related-proteins(GRP78,ATF6,XBP1,e IF2?,ATF4,CHOP,JNK)in liver tissues.The results of this study are as follows:(1)Observation of the superstructure of hepatocytes showed that the nuclear membrane shrinkage and chromatin margination of the IRI and DMEM groups were severe,and the endoplasmic reticulum was severely expanded.After intervention with ADSC-CM or ADSCs,the changes of hepatocyte nuclei and endoplasmic reticulum expansion were slight.(2)Compared with the preoperative level,the relative expression of GRP78,ATF6,IRE1,PERK,ATF4 and CHOP m RNA in liver tissues of the four groups were significantly increased after operation.Compared with IRI group and DMEM group,the intervention with ADSC-CM or ADSCs can significantly reduce the expression of these genes,and there was no significant difference between the two intervention groups.(3)The relative expression of GRP78,ATF6,XBP1,p-e IF2?,ATF4,CHOP and p-JNK protein in liver tissues of the four groups showed an increasing trend.Compared with IRI group and DMEM group,the intervention with ADSC-CM or ADSCs can significantly reduce the expression of these proteins,and there was no significant difference between the two intervention groups.(4)The results of oil red O staining showed that liver cells had severe fatty degeneration and liver cell structures were not clear after operation in the IRI group and DMEM group.The morphology of liver cells in the CM group and ADSCs group was basically normal and the fatty degeneration were mild.(5)Compared with the preoperative level,the levels of TG,CHOL,HDL-C and LDL-C in the serum of the four groups showed a downward trend after operation.Compared with IRI group and DMEM group,the intervention with ADSC-CM or ADSCs can significantly inhibit the reduction of the above indicators in serum.Compared with the preoperative level,the levels of TG,CHOL,HDL,VLDL and ACC1 in the liver of the four groups showed an upward trend after operation.Compared with IRI group and DMEM group,the intervention with ADSC-CM or ADSCs can significantly inhibit the increase of TG,CHOL,HDL,ACC1 content,and significantly increase the VLDL content to maintain the homeostasis of lipid anabolic in the liver.And there were no significant differences in the above indexes in serum and liver between the two intervention groups.(6)Compared with the preoperative level,the relative expression levels of SREBP1,SCAP,FASN,ACC1,HMGCR,HMGCS1,and HMGCS2 m RNA in liver tissue of the four groups were significantly increased after operation.Compared with IRI group and DMEM group,the intervention with ADSC-CM or ADSCs can significantly reduce the expression of the above genes to reduce liver cell lipid synthesis,and there was no significant difference between the two intervention groups.However,the relative expression level of LXR m RNA did not change significantly in different groups and at different time points,indicating that LXR may not participate in the regulation of lipid anabolic in this model.In summary:(1)ADSC-CM can significantly reduce the pathological changes of the ultrastructure of hepatocytes in minipigs,reduce the fat degeneration of hepatocytes,and maintain the normal structure of hepatocytes,indicating that ADSC-CM has protective effects on I/R and partial resection injury in miniature pigs' liver.(2)ADSC-CM can inhibit the overexpression of ERS core protein and three downstream branch pathway signaling molecules,and inhibit the expression of ERS apoptotic pathway.(3)ADSC-CM can inhibit the expression of key regulatory factors and downstream regulatory factors m RNA in hepatocyte lipid synthesis and reduce liver cell lipid deposition.(4)The therapeutic effect of ADSC-CM intervention on hepatocyte ERS and lipid accumulation in the I/R combined partial resection model of miniature pig is basically the same as that of ADSCs,and there is no significant difference between the two. |
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