| Background: Pigmentation is a common skin disease caused by melanin metabolism disorder,which seriously affects the psychological and social activities of patients.The pathogenesis and treatment of this kind of disease has become a research hotspot.Melanin metabolism includes three steps: synthesis,transport and degradation.At present,there are many studies on melanin synthesis and transport,but little is known about the mechanism of melanin degradation.Isoimperatorin(ISO)is a key component in the root of Angelica dahurica,which can inhibit the activity of tyrosinase,an essential enzyme for melanin biosynthesis.However,it is not clear whether ISO affects the degradation of melanin.In this paper,the effect and mechanism of ISO on the degradation of melanin in human keratinocytes were studied.Objective: 1.To verify the regulation of ISO on melanin content;2.To verify the regulation of ISO on the activity and expression of melanin degradation related enzymes;3.To verify that mir3619 targeted to regulate the expression of cathepsin B(CTSB)and cathepsin D(CTSD);4.To clarify that the regulation of ISO on the expression of mi R-3619 affects the activity and expression of CSTB and CSTD.Methods: In the first part,1.Ha Ca T cells were treated with melanosomes and gradient concentrations(0,2,4,8,16,32 ? g / ml)ISO.The viability of Ha Ca T cells was measured at 0,24,48,72 hours by MTT.2.Ha Ca T cells were treated with melanin body and gradient concentration(0,2,4,8 ? g / ml)ISO.The content of melanin was detected by Elisa.3.Ha Ca T cells were treated with melanosomes and gradient concentrations(0,2,4,8 ? g / ml)ISO.The expression of PMEL17 protein was detected by Western Blot(WB).4.Ha Ca T cells were treated with melanosomes and 8 ? g / ml ISO.Alkaline phosphatase,cathepsin B,cathepsin D and cathepsin L2 were detected by biochemical method and Western Blot.In the second part,1.Construct sh RNA interference vectors of CTSB and CTSD genes,and transfect Ha Ca T cells with SH NC,sh CTSB and sh CTSD.Quantitative real time PCR(q PCR)and WB were used to detect the m RNA and protein expression levels of CTSB and CTSD respectively,so as to evaluate the efficiency of SH RNA interference vectors.2.Ha Ca T cells were treated with melanosomes and 8 ? g / ml ISO,then transfected with sh-NC,sh-CTSB and sh-CTSD.The content of melanin was detected by Elisa.3.Ha Ca T cells were treated with melanosomes and 8 ? g / ml ISO,then transfected with sh-NC,sh-CTSB and sh-CTSD.The expression of PMEL17 was detected by WB.In the third part,1.We use the online tool targetscan to predict the mi RNAs that may target CSTB and CSTD at the same time.2.Ha Ca T cells were treated with melanosomes and 8 ? g / ml ISO.The expression level of predicted mi RNA was detected by q PCR.3.The expression of mi R-3619 in Ha Ca T cells was detected by q PCR to evaluate the efficiency of MICs and inhibitor.4.The expression of CSTB and CTSD protein was detected by WB.5.After Ha Ca T cells were treated with melanosomes and 8 ? g / ml ISO,the cells were transfected with mimics NC,mi R-3619 mimics,inhibitor NC and mi R-3619 inhibitor,and the melanin content was detected by Elisa.6.After Ha Ca T cells were treated with melanosome and 8 ? g / ml ISO,the cells were transfected with mimics NC,mi R-3619 mimics,inhibitor NC and mi R-3619 inhibitor,and the expression of PMEL17 was detected by WB.7.Construct the CTSB and CTSD gene 3'UTR luciferase reporter gene vector(wt-CTSB,wt-CTSD),and construct the mutant luciferase reporter gene vector(mut-CTSB,mut-CTSD)by point mutation.293 T cells were co transfected with MIC NC,mi R-3619 mic,inhibitor NC and mi R-3619 inhibitor respectively with wt-CTSB or mut-CTSB to detect luciferase activity;293T cells were co transfected with MIC NC,mi R-3619 mic,inhibitor NC and mi R-3619 inhibitor respectively with wt-CTSD or mut-CTSD to detect luciferase activity.Results: In the first part,1.1,2,4,8 ? g / ml ISO had no effect on the activity of Ha Ca T cells,16,32 ? g / ml ISO reduced the activity of Ha Ca T cells at 48 h and 72 h,indicating that 16,32 ? g / ml ISO affected the growth of cells,which was not suitable for further study.2.1,2 ? g / ml ISO had no effect on the content of melanin in Ha Ca T cells,4,8 ? g / ml ISO reduced the content of melanin in Ha Ca T cells.3.1,2 ? g / ml ISO had no effect on the expression of PMEL17 protein in Ha Ca T cells.4,8 ? g / ml ISO reduced the expression of PMEL17 protein in Ha Ca T cells,indicating that 4,8 ? g / ml ISO was the appropriate concentration.4.8 ? g / ml ISO had no effect on the activity of APC and ctsl2,but increased the activity of CTSB and CTSD.5.8 ? g / ml ISO had no effect on the expression of APC and ctsl2 in Ha Ca T cells,but increased the expression of CTSB and CTSD.In the second part,1.Sh-CTSB and sh-CTSD reduced the m RNA and protein expression of CTSB and CTSD in Ha Ca T cells respectively,which indicated that sh-CTSB and sh-CTSD could be used in subsequent experiments.2.Sh-CTSB and sh-CTSD increased the content of melanin in Ha Ca T cells.3.Sh-CTSB and sh-CTSD increased the expression of PMEL17 protein in Ha Ca T cells.In the third part,1.It is predicted that there are 202 mi RNAs that may target CTSB,49 mi RNAs that may target CTSD,and 16 mi RNAs that may target CTSB and CTSD at the same time.2.ISO increased the expression of mi R-24-3p,mi R-96-5p,mi R-149-5p,mi R-892 b and mi R-661,and decreased the expression of mi R-940,mi R-3619 and mi R-140,among which mi R-3619 decreased the most.3.Mir-3619 mimics increased the expression of mi R-3619 in Ha Ca T cells,and mi R-3619 inhibitor decreased the expression of mi R-3619 in Ha Ca T cells,indicating that mi R-3619 mimics and mi R-3619 inhibitor can be used in subsequent experiments.4.Mir-3619 mimics reduced the expression of CTSB and CTSD in Ha Ca T cells,and mi R-3619 inhibitor increased the expression of CTSB and CTSD in Ha Ca T cells,indicating that mi R-3619 can regulate the expression of CTSB and CTSD.5.Mir-3619 mimics increased the content of melanin in Ha Ca T cells,and mi R-3619 inhibitor reduced the content of melanin in Ha Ca T cells.6.Mir-3619 mimics increased PMEL17 protein expression in Ha Ca T cells,and mi R-3619 inhibitor decreased PMEL17 protein expression in Ha Ca T cells.7.Mir-3619 decreased the luciferase activity of wt-CTSB,but had no effect on mut-CTSB;mi R-3619 inhibitor increased the luciferase activity of wt-CTSB,but had no effect on mut-CTSB,indicating that mi R-3619 targeted to regulate the expression of CTSB gene.Mir-3619 decreased the activity of wt-CTSD luciferase,but had no effect on mut-CTSD;mi R-3619 inhibitor increased the activity of wt-CTSD luciferase,but had no effect on mut-CTSD,indicating that mi R-3619 targeted to regulate the expression of CTSD gene.Conclusion:1.Isoimperatorin significantly reduced the content of melanin in Ha Ca T cells,inhibited the expression of PMEL17,and promoted the activity and expression of cathepsin B and cathepsin D in lysosomes.2.Cathepsin B and cathepsin D can significantly reduce the content of melanin in Ha Ca T cells and inhibit the expression of PMEL17.3.Isoimperatorin significantly inhibited the expression of mir-3619.4.Mir-3619 significantly inhibited the increase of melanin content in Ha Ca T cells,promoted the expression of PMEL17,and inhibited the activity and expression of cathepsin B and cathepsin D in lysosomes.5.Isoimperatorin can reduce the expression of mir-3619,promote the expression of cathepsin B and cathepsin D,reduce the content of melanin in Ha Ca T cells,and inhibit the expression of PMEL17. |
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