Experimental Study On The Mechanism Of Total Flavonoids From Litchi Seed On HSC-T6 Under Different Protein Transfection

Objective:Observe the selected differential proteins(HMOX1,NQO1 and CDK1)transfected with total flavone of Litchi chinensis Sonn(TFL)on the proliferation and inhibition of rat HSC-T6 cells Mechanism,and verify its anti-liver fibrosis effect,provide a basis for its further development and utilization.Methods:The differentially expressed proteins: HMOX1,NQO1 and CDK1 were screened by proteomics and network pharmacology,and the differentially expressed proteins were detected by Western blot.HSC-T6 cells were divided into three groups:(1)blank group(2)model group(3)normal saline group(4)low-dose TFL group(5)medium dose TFL group(6)high-dose TFL group;The experimental groups were as follows:(1)blank control group(2)negative control group(normal saline group)(3)model control group(drug serum group)(4)low-dose litchi nuclear group(5)medium dose litchi nuclear group(6)high-dose litchi nuclear group;(2)negative control group(normal saline group)(3)model control group(drug serum group)(4)low-dose litchi nuclear group(5)medium dose litchi nuclear group(6)high-dose litchi nuclear group;The target protein was selected to transfect hepatic stellate cells,and the m RNA expression level was detected by RT-PCR and Western blot.The experimental groups were as follows:(1)blank control group(2)normal saline group(3)model control group(4)low-dose litchi nuclear group(5)medium dose litchi nuclear group(6)high-dose litchi nuclear group;Flow cytometry was used to detect the changes of apoptosis in each group.The cells were divided into normal group(2)model group(3)HMOX1 overexpression group(4)NQO1overexpression group(5)CDK1 overexpression group(6)HMOX1overexpression + TFL group(7)NQO1 overexpression + TFL group(8)CDK1 overexpression + TFL group;Laser confocal microscopy was used to detect the expression and distribution of the marker proteins,which were divided into:(1)normal group(2)model group(3)HMOX1overexpression group(4)NQO1 overexpression group(5)CDK1overexpression group(6)HMOX1 overexpression + TFL group(7)NQO1 overexpression + TFL group(8)CDK1 overexpression + TFL group;Immunoprecipitation and Western blot were used to detect the interaction between the different proteins,which were divided into:(1)normal group(2)model group(3)HMOX1 overexpression group(4)NQO1 overexpression group(5)CDK1 overexpression group(6)HMOX1 overexpression + TFL group(7)NQO1 overexpression + TFL group(8)CDK1 overexpression + TFL group;Western blot was used to detect the effect of HMOX1,NQO1 and CDK1 overexpression on TGF-β in HSC-T6 β 1、 α-The cells were divided into:(1)blank group(2)model group(3)CDK1 overexpression group(4)CDK1 overexpression+ TFL group(5)HMOX1 overexpression group(6)HMOX1overexpression + TFL group(7)NQO1 overexpression group(8)NQO1overexpression + TFL group.Sppss software 21.0 was used to analyze the correlation between the results of proliferation and inhibition of HSC-T6 by total flavonoids from Litchi seeds transfected with different proteins.Results:(1).Compared with the blank group,the expression of CDK1 in the model group was significantly increased(P<0.05),and the expression of HMOX1 and NQO1 was significantly decreased(P <0.05),and the difference was statistically significant.(2).RT-PCR results showed that the m RNA expression levels of HMOX1,NQO1 and CDK1 were increased in HSC-T6 cells after transfection with HMOX1,NQO1 and CDK1 plasmids(P < 0.05).Western blot showed that the expression levels of HMOX1,NQO1 and CDK1 were increased in HSC-T6 cells after transfection with HMOX1,NQO1 and CDK1 plasmids(P < 0.05).(3).The results of immunofluorescence showed that the expression of HMOX1,NQO1 and CDK1 protein was different in each group after immunofluorescence staining.The expression level of hmox 1 + group was the highest,hmox 1 group decreased slightly;compared with hmox1 group,the expression level of NQO1 + group was lower,compared with NQO1 + group;compared with CDK1 + group,the expression level of CDK1 + group was lower,but there was no significant difference with blank group and model group,the expression level of CDK1 group was the lowest.(4).The results of flow cytometry showed that compared with the blank group,the apoptosis rate of the model group had no significant change,and the CDK1 overexpression group had a certain degree of apoptosis(P < 0.05),The apoptosis rate of CDK1 overexpression + TFL group was significantly increased(P<0.05),the apoptosis rate of HMOX1 overexpression and NQO1 overexpression groups was further increased(P < 0.05),and the apoptosis rate of HMOX1 overexpression+ TFL group and NQO1 overexpression + TFL group was the highest(P< 0.05).(5).The results of immunoprecipitation and Western blot showed that HMOX1,NQO1 and CDK1 proteins had obvious interaction.Moreover,NQO1 and HMOX1,NQO1 and CDK1,HMOX1 and CDK1 interact in NQO1 overexpressed HSC-T6.(6).Western Compared with the model group,the expression of TGF β 1,α-SMA and E-cadherin in CDK1 overexpression group was further increased(P <0.05),CDK1 + TFL was decreased(P<0.05),HMOX1 group and NQO1 group were significantly decreased(P<0.05),HMOX1 + TFL group and NQO1 + TFL group were further decreased(P < 0.05).Conclusion(s): The results showed that: 1.TFN could inhibit liver fibrosis by regulating the differential proteins of HMOX1,NQO1 and CDK1,which was related to the synergistic effect of up regulation of HMOX1,NQO1 and down regulation of CDK1;2.Total flavonoids from litchi seed can regulate the inhibition and proliferation of rat hepatic stellate cells through identified HMOX1,NQO1 and CDK1 differential proteins,and has a certain dose effect relationship;3.Transforming growth factor transfected with different proteins β 1、 α-When SMA and E-cadherin protein were over expressed,HSC-T6 had obvious anti fibrosis effect,and the anti fibrosis effect of E-cadherin protein was more obvious.