Influence Of Total Flavonoids Of Litsea Coreana On The Cytotoxicity Of Oxaliplatin In Testicular Interstitial Cells And Its Possible Mechanism

Objectives:1. To determine the effect of Total Flavonoids of Litsea Coreana (TFLC) on thefunction of gap junction (GJ) and the expression of Cx43in testicular cells.2. To determine the effect of TFLC on the cytotoxicity of oxaliplatin (OHP) intesticular cells (I-10and TM3) and its relationship with GJ.3. To explore the possible mechanisms of TFLC influence the cytotoxicity ofoxaliplatin in testicular cells.Methods:1. Effect of TFLC on GJ in testicular cells1.1Parachute assay was used to detect the effect of TFLC on dye spread of the I-10and TM3cells.1.2Western blotting was used to detect the expression changes of Cx43total proteinin I-10and TM3cells treated with different concentrations of TFLC (0、5、10、20μg/ml) for24h. And immunofluorescence assay was employed to detect theexpression changes of Cx43on the surface of I-10and TM3cells treated withTFLC (0、5、10、20μg/ml) for24h.2. Influence of TFLC on the cytotoxicity of OHP2.1MTT assay was used to detect the effect of TFLC on the cytotoxicity of OHP inconfluent (high density, GJ formed) and pre-confluent (low density, no GJ formed)cells.2.2In order to further verify that the effect of TFLC on the cytotoxicity of OHP inI-10and TM3cells with gap junction, siRNA was designed. After knock-out theCx43of testicular cells, the influence of TFLC on the cytotoxicity of OHP was determined by “MTT assay”.3. Effect of TFLC on the apoptosis induced by OHP3.1“Annexin V/PI double staining” was used to detect the effect of TFLC on theapoptosis of OHP on I-10and TM3cells. The present study is planed to observe theapoptosis of I-10and TM3cells treated with30μM OHP for8h and pretreated with20μg/ml TFLC24h when GJ formed.3.2“Hoechst33258staining” was employed to detect the effect of TFLC on the lateapoptosis of OHP on I-10and TM3cells with pretreated with20μg/ml TFLC for24hwhen GJ formed.4. Effect of TFLC on the Bcl-2, Bax and caspase3/9induced by OHPThe I-10and TM3cells was treated with10μM OHP for24h and pretreated with20μg/ml TFLC24h, the expression of Bcl-2, Bax and caspase3/9was detected bywestern blotting.Results:1. TFLC obviously increased the gap junction function in I-10and TM3cells1.1Parachute assay showed that the dye spread through gap junction in I-10andTM3cells increased when the cells treated with different concentrations of TFLC(0、5、10、20μg/ml) for24h. Cells fluorescence transfer to enhance the respectively20.12%±20.06%（P>0.05）、50.00%±7.30%（P<0.05） and155.00%±13.40%（P<0.01）(vs control group) in I-10cells. Cells fluorescence transfer to enhance therespectively36.50%±2.90%（P<0.05）、85.00%±2.12%（P<0.05） and180.00%±1.41%（P<0.01）(vs control group) in TM3cells. The results showed that TFLCobviously increased the gap junction in I-10and TM3cells.1.2Western blotting was used to detect the expression of Cx43on I-10and TM3cellstreated with TFLC for24h. The results showed the expression of Cx43total proteinincreased with the enhancing of TFLC concentration in I-10and TM3cells.1.3Immunofluorescence assay showed TFLC below20μg/ml significantly enhancedthe expression of Cx43on the surface of I-10and TM3cells. The experimental resultswere the same with the results of western blotting. 2. Effect of TFLC on the cytotoxicity of OHP2.1MTT assay was used to examine the influence of TFLC on the cytotoxicity of OHPin testicular cells. The results showed that20μg/ml TFLC increased the cytotoxicity of10μM OHP in high-density (GJ formed) cells (vs oxaliplatin group, P<0.01), but atlow cell density (no GJ formed), there was no obvious effect of20μg/ml TFLC onOHP cytotoxicity in I-10cells. At same time,20μg/ml TFLC decreased thecytotoxicity of10μM OHP in high-density cells (vs oxaliplatin group, P<0.01), but atlow cell density, there was no obvious effect of20μg/ml TFLC on OHP cytotoxicityin TM3cells.2.2The results showed that the surviving fraction of combination group was noobvious change compared with single-oxaliplatin group after siRNA on Cx43expression in I-10cells, however the surviving fraction of combination groupobviously decreased compared with single-oxaliplatin group in untransfected siRNAcells. The surviving fraction of combination group was no obvious change comparedwith single-oxaliplatin group after siRNA on Cx43expression in TM3cells, howeverthe surviving fraction of combination group obviously increased compared withsingle-oxaliplatin group in untransfected siRNA cells.The results showed that TFLC increased cytotoxicity of OHP via up-regulatingthe function of GJ in I-10cells. However, TFLC decreased cytotoxicity of OHP viaup-regulating the function of GJ in TM3cells.3. Effect of TFLC on the apoptosis induced by OHP3.1Annexin V/PI double staining was used to examine the influence of20μg/mlTFLC on the apoptosis rates of OHP. The results showed that pretreatment of cellswith20μg/ml TFLC for24h, increased the apoptosis rates of30μM OHP inhigh-density cells (vs oxaliplatin group, P<0.05) in I-10cells. Pretreatment of TM3cells with20μg/ml TFLC for24h, decreased the apoptosis rates of30μM OHP inhigh-density cells (vs oxaliplatin group, P<0.05).3.2Hoechst33258staining was used to examine the influence of20μg/ml TFLC onthe late apoptosis rates of OHP, the result showed that, cells pretreated with20μg/mlTFLC for24h, increased the late apoptosis rates of30μM OHP in high-density cells(vs oxaliplatin group, P<0.05) in I-10cells and pretreatment of TM3cells with20μg/ml TFLC for24h, decreased the late apoptosis rates of30μM OHP in high density cells (vs oxaliplatin group, P<0.05).The results showed that TFLC increased the apoptosis rate of OHP in I-10cells.However, TFLC decreased the apoptosis rate of OHP in TM3cells.4. Possible mechanism that TFLC influences the apoptosis induced by OHPWestern blotting showed pretreatment of20μg/ml TFLC significantly enhanced theexpression of Bax, and decreased the expression of Bcl-2in I-10cells (vs oxaliplatingroup, P<0.05). Pretreatment of20μg/ml TFLC significantly decreased the expressionof Bax, and increased the expression of Bcl-2in TM3cells (vs oxaliplatin group,P<0.01). Mitochondrial apoptotic pathway is an important part of the apoptoticprocess. The experiment also detected the expression of caspase3/9. Western blottingresults showed the cleaved caspase3/9of combination group was significantlyincreased in I-10cells comparing with single-oxaliplatin group; the cleavedcaspase3/9of combination group was obviously decreased in TM3cells comparingwith single-oxaliplatin group.In total, TFLC increased the cytotoxicity of OHP by enhancing the expression ofcleaved caspase3/9and upregulating the expression of Bax/Bcl-2in I-10cells. TFLCdecreased the cytotoxicity of OHP by inhibiting the expression of cleaved caspase3/9and downregulating the expression of Bax/Bcl-2in TM3cells.Conclusions：1. TFLC increased the function of GJ and the expression of Cx43in I-10and TM3cells.2. TFLC increased the cytotoxicity of OHP on I-10cells, however, decreased thecytotoxicity of OHP on TM3cells.3. TFLC increased the apoptosis induced by OHP on I-10cells, however, decreasedthe apoptosis induced by OHP on TM3cells.4. Bcl-2/Bax and caspase3/9maybe involved in the effect of GJ on the OHPcytotoxicitity.