The Regulation Effect Of Duhuojisheng Decoction On Zinc-ZIP8-MTF1 Axis In Mice Cartilage Cell

Objective:Cartilage cells in mice by living by parasitic soup any Zinc-ZIP8-MTF1 axis regulation function research,explore the living by parasitic soup treatment of Knee Osteoarthritis,Knee Osteoarthritis,KOA)targets and functions of laws,reveals the treatment mechanism.To provide reliable experimental basis for the treatment of Knee Osteoarthritis?KOA?.Method:The 20 SD rats were randomly divided into two groups:blank control group and experimental group,each group of 10.The single live parasitic soup was administered by 10 times of the clinical equivalent dose,and the blank control group was given normal saline,and the stomach was filled with 1mL/100g weight.1 time in the morning,afternoon and a day for 3 days,after the last for 1 h,the abdominal aorta in blood,serum,centrifugal separation and 56?water bath 30 min inactivated,repackaging,-80?cryopreserved,live with parasitic soup drug-containing serum standby,the express Zip8 adenovirus infection Ad-Zip8?Vector Biolabs?infected mice chondrocytes,infection of the plural is 800 moi,infection time is 2 hours,24hours continue to develop to build Zip8 expression cell models used in the experiment.To eliminate the interference of multi-target effect of traditional Chinese medicine,the expression of siRNA targeted silence Zip8 was used to construct the zip8-sirna gene silencing cell model for the experiment.Cells were randomly divided into:the blank Control group?Control?,Model Control group?Model/ZnCl2?,Ad-Zip8 group?Ad-Zip8?,Zip8-siRNA group?Zip8-siRNA?DHJST?2.5%?,low dose group,middle dose group?5%?DHJST DHJST?10%?,high dose group,each group after three holes,grouping,training system to join 1 ng/mL IL-1 beta;After 24 h cultivation,100uM ZnCl₂ was added,and the drug containing serum was administered.The drug containing serum of 10%,5%and 2.5%?v/v?were used as high,medium and low dose groups?10%of the dose group with the drug serum,and 10%of the normal rat serum?;10%normal rat serum was used as a control group.Continue to develop after48 h using fluorescence staining techniques to detect cartilage cell Zn²⁺concentration,the total RNA extracted cells,remove the gDNA,synthesis of cDNA,the qRT-PCR detection of chondrocytes ZIP8,ZNT9,MTF1,Mt1 and Mt2 gene expression,protein immunoblot method?Western Blot?combined with image analysis ZIP8,ZNT9,MTF1,Mt1 and Mt2 protein expression.Results:1.Chondrocytes in the condition of IL-1?induced inflammation,cell Zn²⁺concentration significantly increased,living by join parasitic soup after medicated serum?DHJST?has certain influence to the Zn²⁺concentration,dyeing imaging by the picture visible drug-containing serum low dose group of Zn²⁺concentration is reduced,reduce dyeing part,5%of the medicated serum middle dose group of Zn²⁺concentration,10%of the lowest drug-containing serum high dose group of Zn²⁺concentration,thus it can be seen live with parasitic soup drug-containing serum?DHJST?the higher the concentration,the concentration of Zn²⁺is lower,dyeing imaging part is less,the more light,color alone live parasite soup drug-containing serum?DHJST?for Ad-Zip8 overexpression of Zn²⁺concentration within the cell model has a role in reducing.The serum?DHJST?of single live parasitic soup had a certain reduction in the concentration of Zn²⁺in the cell model of zip8-sirna gene.To sum up,living by parasitic soup drug-containing serum?DHJST?on intracellular concentration of Zn²⁺has certain inhibition,living by parasitic soup drug-containing serum?DHJST?content is higher,the cell Zn²⁺concentration is lower,on the other hand,the higher the Zn²⁺concentration in the cell,so live alone parasitic soup drug-containing serum?DHJST?can decrease the inflammatory state of cartilage cell Zn²⁺concentration.2.According to the results of rt-qpcr,the serum of single live parasitic soup had obvious inhibitory effect on the expression level of Zip8 mRNA,but its effect was less than that of zip8-sirna.Also found that living by parasitic soup drug-containing serum no match for the Ad-Zip8 trigger Zip8 mRNA expression,living by parasitic soup medicated serum of high zinc state of chondrocytes Znt9 mRNA expression level without significant impact,but found that caused by Ad-Zip8 Zip8 ever mRNA expression can cause Znt9 mRNA expression levels,this phenomenon may be associated with Zn²⁺influx result in higher Znt9 negative feedback,living by parasitic soup drug-containing serum can cut Mtf1 mRNA expression level,but its strength is less than the Mtf1-siRNA.Live with parasitic soup drug-containing serum can be effective against Ad-Zip8 trigger Mtf1 mRNA expression levels,live with parasitic soup medicated serum could significantly increase Mt1 mRNA expression level,and the Mt1 mRNA expression level raised effect from Zip8 mRNA expression level.3.According to Western blotting,the expression level of ZIP8 protein can be reduced by the serum of single live parasitic soup,but its effect is less than that of zip8-sirna.Live with parasitic soup medicated serum could not inhibit triggered by Ad-Zip8 Zip8 protein expression levels,live with parasitic soup medicated serum of high zinc ZNT9 protein expression level of cartilage cells under no significant effect,Ad-Zip8 can result in higher ZNT9 protein expression level of feedback alone live parasite soup drug-containing serum protein expression level had no obvious effect on MTF1,the results are not consistent with the result qPCR.Live with parasitic soup drug-containing serum can effectively reduce the Ad-Zip8 MTF1 caused by the protein expression level,living by parasitic soup drug-containing serum can obviously increase the expression of MT protein levels,and its effect on MT protein expression level of increase is not affected by Zip8 had expressed.Conclusion:1.The concentration of Zn²⁺in the cells with inflammatory state was decreased by the serum?DHJST?,and the higher the serum content,the stronger the reduction of Zn²⁺concentration.2.The expression level of ZIP8 protein can be reduced by the serum of single live parasitic soup,which has obvious inhibitory effect on the expression level of zip8-mrna,but can't resist the expression of ZIP8 mRNA caused by ad-zip8.Live with parasitic soup medicated serum of high zinc state of chondrocytes Znt9 mRNA expression level without significant impact,but found that caused by Ad-Zip8 Zip8ever mRNA expression can cause Znt9 mRNA expression levels,this phenomenon may be related to Zn²⁺influx result in higher Znt9 negative feedback.3.Live with parasitic soup drug-containing serum protein expression level had no obvious effect on MTF1,but live alone parasitic soup drug-containing serum can effectively reduce the Ad-Zip8 MTF1 caused by the protein expression level,also can obviously increase the expression of MT protein levels,and its effect on MT protein expression level of increase is not affected by Zip8 had expressed.4.Live with parasitic soup can reduce the concentration of Zn²⁺,reduce the Zn²⁺transporters especially ZIP8 protein levels,decrease MTF1 activated,reduce the cartilage cells the expression of MMPS family and ADAMTS family,for the living by exploring the parasitic soup treatment of KOA targets and work rules,reveal its action mechanism is of great significance.