Effect Of Total Flavonoids Of Semen Litchi On Expression Of Pathological Related Factors In Hepatic Fibrosis

Objective:To investigate the effect of total flavonoids of semen litchi（TFL）in the pathological process of hepatic fibrosis（HF）on the apoptosis of human hepatic stellate cells（HSC-LX2）induced by transforming growth factor-β₁（TGF-β₁）and the expression of TLR4,NF-κB and IL-1RⅠ.Methods:The cultured HSC-LX2 was randomly divided into different groups including normal group,TGF-β₁ group and different concentrations of TFL groups（150,300 and 600mg/L TFL）.TGF-β₁ group and different concentrations of TFL groups cells were cultured in the 10 cm culture dish,the dish containing DMEM（10%FBS）and TGF-β₁（5μg/L）,after 24 h culture,different concentrations of TFL groups were added to different concentrations of TFL（150,300 and600mg/L TFL）,After 48 h of culture,related indicators were measured.Observation of cell morphology by Hoechst33258 staining;Cell cycle was detected by flow cytometry;Apoptosis was detected by FITC Annexin V/PI Double staining;The expression levels of TLR4,NF-κB and IL-1RⅠprotein were detected by Western blot assay.Results:（1）Hoechst33258 stainin showed that the normal group and the TGF-β₁ group of nuclear morphology complete;morphological changes of apoptosis including pyknosis,lysis and apoptotic bodies of nucleus were observed in different concentrations of TFL groups（150,300 and 600mg/L TFL）.（2）Flow cytometry showed that the percentage of G0/G1 phase cells（45.56±0.72）and S phase cells（45.21±0.18）in the TGF-β₁group and the percentage of G0/G1 phase cells（53.74±0.96）and S phase cells（34.93±1.89）in the normal group had obvious difference（P<0.05）.Compared with the TGF-β₁ group,the 150mg/L TFL group both the G0/G1 phase cells percentage（58.05±5.17）and S phase cells percentage（33.47±4.67）,the300mg/L TFL group both the G0/G1 phase cells percentage（73.23±4.08）and S phase cells percentage（19.97±3.88）,600mg/L TFL group both the G0/G1 phase cells percentage（86.91±2.15）and S phase cells percentage（7.01±1.54）in the G0/G1 phase cells percentage and S phase cells percentage respectively had obvious difference（P<0.05）.With the increasing of TFL concentration,G0/G1 phase cells increased（P<0.05）,S phase cells decreased（P<0.05）,TFL blocked cells in G0/G1 phase.Early apoptosis rate（1.16±0.60）,late apoptosis rate（5.53±2.41）and total apoptosis rate（6.70±3.01）in the TGF-β₁ group were not significantly different from early apoptosis rate（1.03±0.75）,late apoptosis rate（6.80±3.34）and total apoptosis rate（7.83±3.26）in the normal group（P>0.05）;There were significant difference in early apoptosis rate,late apoptosis rate and total apoptosis rate between TGF-β₁ group and 150mg/L TFL group（early apoptosis rate（18.16±6.73）,late apoptosis rate（14.93±0.80）and total apoptosis rate（33.10±6.75））（P<0.05）;Compared with the TGF-β₁ group,both the300mg/L TFL group early apoptosis rate（25.30±1.57）,late apoptosis rate（15.80±0.43）,total apoptosis rate（41.13±1.15）and the 600mg/L TFL group early apoptosis rate（18.96±1.36）,late apoptosis rate（29.53±1.16）,total apoptosis rate（48.50±1.13）in the early apoptosis rate,late apoptosis rate,total apoptosis rate respectively had obvious difference（P<0.05）.With the increasing of TFL concentration,TFL can promote the apoptosis of HSC-LX2,tend to promote late apoptotic.（3）The Western blot assay showed that the expression levels of TLR4 protein（1.05±0.11）,NF-κB protein（1.19±0.10）and IL-1RⅠprotein（1.17±0.12）in the TGF-β₁ group and the expression levels of TLR4protein（0.86±0.74）,NF-κB protein（0.88±0.13）and IL-1RⅠprotein（0.90±0.12）in the normal group had obvious difference（P<0.05）.Compared with the TGF-β₁ group,in the 150mg/L TFL group the expression levels of TLR4 protein（0.87±0.05）,NF-κB protein（0.77±0.05）and IL-1RⅠprotein（0.78±0.02）,300mg/L TFL group the expression levels of TLR4 protein（0.61±0.05）,NF-κB protein（0.56±0.07）and IL-1RⅠprotein（0.53±0.08）,600mg/L TFL group the expression levels of TLR4 protein（0.40±0.07）,NF-κB protein（0.36±0.06）and IL-1RⅠprotein（0.35±0.05）in the expression levels of TLR4 protein,NF-κB protein and IL-1RⅠprotein respectively had obvious difference（P<0.05）.In the different concentrations of TFL groups（150,300 and 600mg/L TFL）,TLR4 protein,NF-κB protein and IL-1RⅠprotein expression levels were lower than TGF-β₁ group（P<0.05）;and with the increase of TFL concentration,the expression levels of TLR4 protein,NF-κB protein and IL-1R I protein in HSC-LX2 decreased gradually.Conclusions:（1）The anti-hepatic fibrosis mechanism ofTFL may be related to TFL promotes the apoptosis of HSC-LX2 induced by TGF-β₁;（2）The molecular mechanism of TFL promoting HSC-LX2 apoptosis induced by TGF-β₁ may be related to the reduction of the expression of TLR4 and NF-κB in HSC-LX2;（3）TLR-IL-1R inflammatory pathway may be involved in the process of hepatic fibrosis,and it is expected to be a new way of anti-hepatic fibrosis by inhibiting the inflammatory pathway of TLR-IL-1R.