Investigation Of The Mechanism Of Cdc37/Akt Signaling Pathway In The Proliferation And Apoptosis Of Keratinocytes And The Effect Of Shikonin

Objective:Accelerated proliferation and reduced apoptosis of epidermal keratinocytes,shortened mitotic cycle and epidermal turnover time are characteristic manifestations of psoriasis.Studies have shown that keratinocytes are not only involved in the initiation of psoriasis,but also interact with immune cells in the further development of psoriasis and the relapse process.Therefore,exploring the mechanism and inhibition strategy of abnormal proliferation of keratinocytes and achieve clinical translation will be valuable for psoriasis treatment.Cell division cycle 37(Cdc37)has chaperone molecular activity and specifically mediates the binding of multiple protein kinases to heat shock protein(HSP)90,forming a specific molecular chaperone complex that protects protein kinases from degradation and plays a significant role in cell cycle,signal transduction and gene expression.It has been shown that Cdc37 expression is elevated in a variety of malignant neoplasm tissues and in some benign diseases.However,the expression and mechanism of action of Cdc37 in psoriatic keratinocytes have not been reported.Therefore,this study will first verify the expression of Cdc37 in skin lesions of psoriasis patients by database search and immunohistochemical assay of clinical samples;Then we will further observe the effect of Cdc37 expression alteration on proliferation,cell cycle and apoptosis of keratinocytes;Finally,we will investigate the potential molecular mechanism of Cdc37 function.Zicao is used in clinical as a Chinese herbal formula for the treatment of psoriasis.Shikonin is the main active ingredient of Zicao,which has various biological activities such as antiinflammatory,anti-viral and anti-tumor.Several basic studies have confirmed the possible mechanism of its effect on psoriasis,but there is no clinical use of shikonin to treat psoriasis.More basic researches are still needed to provide the evidences for the effectiveness and safety of its clinical application.We have discovered in previous studies that shikonin inhibited keratinocytes proliferation and induced apoptosis.It has also been shown that shikonin regulated Cyclin A,Cyclin B,Cyclin D,Cyclin E,CDK1,CDK2 and other cyclerelated proteins that interact with Cdc37 to inhibit the proliferation of tumor cells.However,there are no studies on the association of shikonin with cell cycle-related factors in psoriatic keratinocytes.This study will explore whether shikonin affects the abnormal alteration of keratin-forming cells induced by Cdc37 both in vivo and in vitro,and explore the possible targets of action in an attempt to provide important scientific data for the elucidation of the pathogenesis of psoriasis and the clinical application of shikonin.Methods:1.The GEO database was retrieved to obtain datasets of the expression of Cdc37 m RNA between psoriasis patients and healthy controls.Datasets with organisms other than homo sapiens,cases with few samples or cases irrelevant to psoriasis were excluded.Paraffin blocks were collected after clinicopathological diagnosis,and immunohistochemical staining was used to observe the differences in the expression of Cdc37 protein levels in psoriatic lesions and controls,as well as the differences in its distribution in the various layers of the skin.2.Cdc37 overexpression cell model LV-Cdc37 was constructed by lentiviral transfection,and the transfection efficiency was verified by RT-q PCR and Western blot;cellular models of Cdc37-HSP90 complex inhibition were constructed with Hsp90 inhibitor 17-AAG.Based on the successful construction of cell models,the cell proliferation rate was detected by CCK-8 and the proportion of cells in proliferative phase was detected by the Ed U assay;The changes of cell cycle distribution and apoptosis between different groups were detected by flow cytometry.Combining the signaling pathway mapping in the KEGG database and related research advances in psoriasis,the expression differences of pathway molecules were verified by Western blot technique.3.Clarification whether shikonin affects the role of Cdc37/Akt in keratinocytes: The effect of shikonin on the proliferation rate of LV-Cdc37 cells was detected by CCK-8;The proportion of LV-Cdc37 cells in proliferative stage was detected by Ed U method;The changes of cell cycle and apoptosis of LV-Cdc37 cells after shikonin intervention were detected by flow cytometry;The expression changes of related protein molecules were verified by Western blot.Psoriatic mouse model induced by imiquimod was used to verify the effect of shikonin on psoriatic lesions;The expression changes of related protein molecules in animal models were verified by Western blot.Results:1.Two eligible datasets were retrieved from the GEO database,and the statistical results both showed that the expression of Cdc37 m RNA was significantly increased in psoriasis patients’ skin lesions compared with that in non-lesioned and normal human skin tissues,and no significant difference was seen in the expression of Cdc37 m RNA in psoriasis patients’ non-lesioned and normal human skin tissues.Immunohistochemical assays of skin tissues from psoriasis patients and normal controls showed that Cdc37 was distributed to the basal cell layer of the epidermis in healthy skin,whereas in the lesions of psoriasis patients,Cdc37 was expressed not only in the basal layer but also in the spiny layer cells.The results quantified with Image Pro-Plus 6.0 software also suggested that the expression of Cdc37 was significantly higher in the skin lesions of psoriasis patients than in the control group.2.The results of CCK-8 assay showed that LV-Cdc37 cells proliferated significantly faster from day 3 compared with Ha Ca T cells,while 17-AAG inhibited the proliferation rate of LV-Cdc37 cells.The results of Ed U assay showed that the proportion of Ed U-positive cells in LV-Cdc37 cells was significantly higher,representing a significant increase in the proportion of cells in proliferative phase,while 17-AAG reduced the proportion of cells in proliferative phase in LV-Cdc37 cells.After flow cytometric detection of the cell cycle,the results showed that the proportion of cells in the G1 phase was reduced and the proportion of cells in both the S and G2/M phases was increased in LV-Cdc37 cells compared with EV cells;the proportion of cells in the G1 phase was increased and the proportion of cells in both the S and G2/M phases was reduced in LV-Cdc37 cells after 72 h of 17-AAG intervention at a concentration of 10 n M.The results of apoptosis detection by flow cytometry showed that the proportion of apoptotic cells was significantly reduced in LVCdc37 cells compared with EV cells;17-AAG caused a significant increase in the proportion of apoptotic cells in Ha Ca T cells.The results of Western Blot showed that compared with EV cells,p-Akt,Cyclin D1,Bcl-x L expression was elevated and BAD protein expression was reduced in LV-Cdc37 cells,while Akt,p-ERK1/2,CDK4/6,Cyclin D2,Cyclin D3,Bcl-2 and C-casp-9 proteins were not changes.In order to clarify the key regulatory point,MK2206,an inhibitor of Akt phosphorylation site,was used to co-culture with LV-Cdc37 cells,and then Western blot was used to detect changes in the expression of related proteins again,which showed that the expression of p-Akt,Cyclin D1 and Bclx L was reduced and the expression of BAD was increased.3.Effect of shikonin on Cdc37/Akt signaling pathway: Treatment of LV-Cdc37 cells with shikonin(0.5μM or 1μM)for 72 h reduced the proliferative activity of LV-Cdc37 cells,increased the proportion of G1-phase cells and reduced the proportion of both S-phase and G2/M-phase cells in LV-Cdc37 cells,and significantly increased the proportion of apoptotic cells in LV-Cdc37.The results of Western Blot showed that shikonin did not affect the expression of Cdc37,but significantly decreased the protein expression of Akt and downstream p-Akt,Cyclin D1,Bcl-x L in concentration-dependent manner,and significantly increased the expression of BAD protein.In vivo experiments showed that shikonin significantly improved imiquimod-induced skin lesion in mice,and Western Blot showed that shikonin played a role by reducing the expression of Akt protein and reducing its phosphorylation.Conclusion: 1.Cdc37 expression is elevated in skin lesions of psoriasis patients compared to normal controls.2.With Akt as the key regulatory point,Cdc37 promoted the phosphorylation of Akt in keratinocytes,further increased the expression of Cyclin D1 and differentially regulated the expression of Bcl-2 family proteins(Bcl-x L and BAD),then accelerated the proliferation of keratinocytes,inhibited the apoptosis of keratinocytes,and promoted the transition of keratinocytes from G1 phase to S phase,and these effects were accomplished jointly by binding to HSP90.3.Shikonin reduced the expression of Akt protein,decreased its phosphorylation,and then regulated the expression of downstream protein molecules Cyclin D1,Bcl-x L and BAD,which inhibited the accelerated proliferation of keratinocytes induced by Cdc37,reduced the proportion of cells transitioning from G1 to S phase,and induced apoptosis,ultimately improving the severity of psoriatic lesions in mice.