| Objectives:Cervical cancer has the characteristics of high morbidity,high mortality,and younger age of onset,which seriously threatens women's physical and mental health.In recent years,the incidence of cervical cancer in China has been on the rise.As a public health issue,cervical cancer prevention and treatment has attracted great attention and concern from the Chinese government.Among many treatments for cervical cancer,photodynamic therapy(PDT)is a new type of therapy with broad prospects in tumor treatment.It is a non-invasive or minimally invasive treatment technology that combines light,photosensitizers and oxygen molecules in tissues,selectively destroys pathological tissues through photodynamic reaction to achieve the purpose of treatment.It has small trauma and toxic side effects on pathological parts,precise positioning,short treatment time and operation with the advantages of simplicity and low price;it has a broad prospect in tumor treatment.For most tissues,the depth of PDT can meet the treatment needs,while the superficial part of cervix is easy to be exposed.Local administration and light treatment are very convenient,which makes PDT more advantageous in the treatment of cervical lesions.In addition,PDT can carry out repeated treatment for many times on the premise of preserving the complete organ,and remove the residual pathological tissue,so as to thoroughly cure the pathological changes with the diameter of more than 3cm and the depth of involving the cervical gland cavity more than 2.5cm,which highlights the treatment advantage.At the same time,after PDT treatment,women's reproductive function has not been damaged,which can meet the requirements of young patients to retain fertility.The key to PDT treatment depends on the use of ideal photosensitizers.The previous generations of photosensitizers used in clinics are complex and easily affected by biological metabolism.The degree of photosensitivity damage is not easy to control,and toxic side effects and tolerability will occur after repeated use,thus limiting the effectiveness of PDT treatment.At present,a new generation of photosensitizers is under development,and manyChinese medicine monomers(such as curcumin,baicalin,etc.)have good photosensitivity.The team conducted research on curcumin-mediated PDT therapy for cervical cancer in the early stage of Chinese medicine,and confirmed that curcumin-mediated PDT has a certain effect on cervical cancer.Baicalin has obvious antiviral,anti-inflammatory,anti-tumor,and treatment-related gynecological diseases.Therefore,we suspect that its combination with PDT for cervical cancer will have better efficacy than curcumin,and it is expected to become an efficient,low-toxic and new photosensitizer with multiple anti-tumor effectsTherefore,this experiment intends to use baicalin mediated PDT therapy in the treatment of cervical cancer,to observe the inhibitory effect of this therapy on tumor tissue,and to explore whether its possible mechanism of tumor inhibition is related to the promotion of cancer cell apoptosis.The experiment is divided into two parts.Experiment 1:establish the tumor model of cervical cancer transplantation in vitro,and give the corresponding treatment.Through the change of tumor volume and the comparison of tumor inhibition rate of each group,the therapeutic effect of baicalin mediated PDT on cervical cancer can be observed,and the optimal concentration of Baicalin can be filtered;the therapeutic effect of the treatment from the histopathological changes can be further verified through HE staining.Experiment 2:to further explore the mechanism of baicalin mediated PDT therapy in the treatment of cervical cancer transplanted in vitro.In order to investigate whether the apoptosis mechanism of baicalin mediated photodynamic therapy for cervical cancer in vitro is related to the Bcl-2/Bax/Cyt-C pathway,immunohistochemistry and Western blot were used to detect the distribution and expression of Bcl-2,Bax and Cyt-C proteins.Methods:1.Observation of the therapeutic effect of baicalin mediated photodynamic therapy on cervical cancer transplanted in vitro and selection of the best concentration.HeLa cells were inoculated subcutaneously on the back of BALB/c nude mice to establish a model of cervical cancer transplantation in vitro.The tumor bearing mice were randomly and evenly divided into model group,high concentration baicalin-PDT treatment group,medium concentration baicalin-PDT treatment group,low concentration baicalin-PDT treatment group,curcumin-PDT treatment group and PDT treatment group,12 in each group.PDT group:100?L normal saline was injected around the tumor,and O.Olmol/L borax buffer was injected around the tumor 6 hours later.PDT was performed at a dose of 80J/cm2(435nm,0.60A,0.54W,3-5cm from the tumor surface,2min28s).;high,medium and low concentration baicalin-PDT treatment group:baicalin solution of different concentrations of 80?g/mL,60?g/mL and 40 ?g/mL was used instead of normal saline,the other was the same as PDT treatment group;curcumin-PDT treatment group:curcumin solution of 50 ?mol/L was used instead of normal saline,the other was the same as PDT treatment group;model group:natural light was used instead of PDT treatment,the other was the same as PDT treatment group.After 1 day of treatment,6 nude mice in each group were randomly selected to make paraffin sections,and the histopathological changes were observed by HE staining.The other six nude mice were measured on the first;third,seventh and fourteenth day of treatment respectively,and the tumor volume inhibition rate was calculated to determine the therapeutic effect of baicalin mediated PDT on cervical cancer in vitro.2.Study on the mechanism of baicalin mediated photodynamic therapy in the treatment of cervical cancer.HeLa cells were inoculated subcutaneously on the back of BALB/c nude mice to establish a model of cervical cancer transplantation in vitro.The tumor bearing mice were randomly and evenly divided into model group,baicalin-PDT treatment group,PDT treatment group and curcumin-PDT treatment group,there were 12 animals in each group PDT group:100?L normal saline was injected around the tumor,and 0.01 mol/L borax buffer was injected around the tumor 6 hours later.PDT was performed at a dose of 80J/cm2(435nm,0.60A,0.54W,3-5cm from the tumor surface,2min28s);baicalin-PDT treatment group:baicalin solution of the optimal concentration selected(80?g/mL)in Experiment 1 was used to replace normal saline,the other was the same as PDT treatment group;curcumin-PDT treatment group:Curcumin solution of 50?mol/L was used to replace normal saline,the other was the same as PDT treatment group;model group:natural light was used to replace PDT treatment,the other was the same as PDT treatment group.Immunohistochemistry and Western blot were used to detect the distribution and expression of Bcl-2,Bax and Cyt-C protein in the transplanted tumor tissues,and to explore whether the apoptosis mechanism of baicalin mediated photodynamic therapy for cervical cancer in vitro is related to the apoptosis pathway of Bcl-2/Bax/Cyt-C.Results1.Baicalin mediated photodynamic therapy has therapeutic effect on cervical cancer transplanted in vitro,among which high concentration baicalin(80?g/mL)is the best.The effect is better than curcumin mediated photodynamic therapy group1.1 Results of tumor volume changesIn the model group,the tumor volume increased gradually.The tumor growth of each treatment group was inhibited to varying degrees,and the volume remained unchanged or slightly decreased from 1 to 3 days after treatment,and gradually increased after 3 days;compared with the model group,the tumor volume of high concentration baicalin PDT treatment group and curcumin PDT treatment group decreased significantly after 14 days of treatment,the difference was statistically significant(P<0.01),and the tumor volume of other treatment groups decreased,But there was no statistical difference(P>0.05).Compared with PDT treatment group,the tumor volume of high concentration baicalin PDT treatment group decreased significantly after the 14th day of treatment,the difference was statistically significant(P<0.05),the tumor volume of other treatment groups decreased,but there was no statistical difference(P>0.05).1.2 Results of changes in tumor volume inhibition rate1-3 days after treatment,the inhibition rate of high concentration baicalin PDT treatment group and curcumin PDT treatment group increased,and the inhibition effect of high concentration baicalin PDT treatment group was significantly better than curcumin PDT treatment group,and the inhibition rate of other treatment groups showed a downward trend;7-14 days after treatment,the inhibition rate of each group showed a downward trend,but the inhibition rate of high concentration baicalin PDT treatment group was higher than that of curcumin PDT treatment group Other groups.1.3 Histopathological resultsUnder the light microscope,it can be seen that the tumor cells in the model group are relatively single,arranged in the shape of mass nests,with different cell sizes,unclear borders,round or oval shape,increased nucleoplasma ratio,rough chromatin,common mitotic images,and occasional necrosis.Compared with the model group,the rest of the treatment groups showed necrosis,nuclear pyknosis,eosinophilic change of cytoplasm,sparse arrangement of tumor cells around the necrosis,reduced volume,and decreased mitotic image compared with the model group,especially in the high concentration baicalin PDT treatment group,curcumin PDT treatment group.In addition,the necrosis area of tumor tissue in high concentration baicalin PDT treatment group was larger than that in other treatment groups,while there was bleeding in the tumor tissue in curcumin PDT treatment group.2.The apoptosis mechanism of baicalin mediated photodynamic therapy is related to Bcl-2/Bax/Cyt-C pathway.2.1 Immunohistochemical results:The expression of Bcl-2,Bax and Cyt-C protein were all distributed in the cytoplasm of the cells,which was brown yellow2.1.1 Bcl-2:After 1 day of treatment,compared with the model group,the expression of Bcl-2 in the baicalin-PDT treatment group,curcumin-PDT treatment group and PDT treatment group were significantly reduced(P<0.01);compared with curcumin-PDT treatment group,the expression of Bcl-2 in the baicalin-PDT treatment group had no statistical difference(P>0.05).2.1.2 Bax:After 1 day of treatment,compared with the model group,the expression of Bax in the baicalin-PDT treatment group and curcumin-PDT treatment group increased significantly(P<0.01),and Bax expression in the PDT treatment group increased(P<0.05);compared with the curcumin-PDT treatment group,Bax expression in the baicalin-PDT treatment group had no statistical difference(P>0.05).2.1.3 Cyt-C:After 1 day treatment,compared with the model group,the expression of Cyt-C in the baicalin-PDT treatment group,curcumin-PDT treatment group and PDT treatment group increased significantly(P<0.01);compared with curcumin PDT treatment group,the expression of Cyt-C in baicalin-PDT treatment group had no statistical difference(P>0.05).2.2 Western blot results:2.2.1 Bcl-2;After 1 day of treatment,compared with the model group,the expression of Bcl-2 in the baicalin-PDT treatment group and the curcumin-PDT treatment group was significantly reduced(P<0.01),while that in the PDT treatment group was reduced.(P<0.05);Compared with the curcumin-PDT treatment group,the expression of Bcl-2 in the baicalin-PDT treatment group was no statistical difference(P>0.05).2.2.2 Bax:After 1 day of treatment,compared with the model group,the expression of Bax in the baicalin-PDT treatment group and curcumin-PDT treatment group significantly increased(P<0.01),and there was no statistical difference in Bax expression in the PDT treatment group(P>0.05).Compared with the curcumin-PDT treatment group,there was no statistical difference in Bax expression in the baicalin-PDT treatment group(P>0.05),2.2.3 Cyt-C:After 1 day of treatment,compared with the model group,the expression of Cyt-C in the baicalin-PDT treatment group,curcumin-PDT treatment group and PDT treatment group increased significantly(P<0.01);compared with curcumin-PDT treatment group,the expression of Cyt-C in baicalin-PDT treatment group had no statistical difference(P>0.05).Conclusion1.Baicalin mediated photodynamic therapy can inhibit cervical cancer cells.High concentration baicalin mediated photodynamic therapy is the best,and its overall effect is better than curcumin mediated photodynamic therapy;2.The inhibitory effect of baicalin mediated photodynamic therapy on cervical cancer transplanted in vitro may be related to Bcl-2/Bax/Cyt-C related apoptosis pathway. |
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