Study On The Mechanism Of Hypothermia Inhibiting The Hair Follicle Injury Induced By Paclitaxel

In cancer treatment,chemotherapy-induced alopecia（CIA）is one of the intolerable side effects,which may bring serious mental health problems to patients.Based on the urgent needs of clinical patients,the prevention of chemotherapeutic alopecia has always been one of the important tasks in the process of cancer treatment.The clinical report pointed out that scalp cooling technology has great development potential for preventing hair loss during chemotherapy,and can be used as an effective clinical adjuvant treatment.Scalp cooling can induce scalp vasoconstriction through physical cooling of scalp,reduce drug diffusion rate,and reduce the concentration of chemotherapy drugs entering scalp hair follicles.However,most studies at this stage analyzed the mechanism of scalp cooling from each single factor,without considering the synergistic effect of multiple factors,and without analyzing the protective mechanism of scalp cooling from the molecular level.Based on this,the problems of scalp cooling were systematically explored to prevent CIA in this paper.Through cell biology and in vitro experiments,the target genes for hypothermia were screened,the toxicology of paclitaxel on hair follicles in vitro was studied,and the biological mechanism of hypothermia inhibiting paclitaxel induced chemotherapeutic alopecia injury by combining the theoretical analysis of mass transfer and drug generation of paclitaxel was explored.The main contents and conclusions are as follows:（1）The synergistic effect of hypothermia and taxol concentration on cell growth was studied by constructing the cytotoxicity model induced by taxol in vitro,epidermal keratinocytes（NHEK）and mouse hair follicle keratinocytes（MHFK）were considered.The effects of different temperatures on cell growth and chemotherapy with different concentrations of paclitaxel were investigated.The results showed that the lower the temperature was,the slower the cell proliferation was.At 22℃,the process of cell mitosis was very slow,and the generation duration was 3.1 times longer.The toxicity of paclitaxel to both kinds of cells is dose dependent.The lower the temperature,the more obvious the reduction of cytotoxicity.The results of cell cycle showed that pacxlitaxel will block the cell cycle in G2/M phase.Hypothermia can reduce the impact of taxol on the cell cycle,balance the cell cycle,and finally restore the cell cycle to normal.Paclitaxel induces mitochondrial transmembrane potential（ΔΨm）Fracture,mild hypothermia can transform cells fromΔΨm breaks,stabilize mitochondrial membrane potential,reduce ROS of cells and reduce apoptosis.During the cooling process,the expression of heat shock protein family 70（HSP70）was significantly up-regulated,and the concentration was 3 times of the original.（2）Based on bioinformatics analysis method,RNA seq was sequenced on NHEK cells in chemotherapy group and mild hypothermia treatment group to clarify the molecular mechanism of mild hypothermia on paclitaxel induced cell damage protection at the cellular level.The results showed that hypothermia treatment mainly affected HSPA1A,HSPA1B and HSPA8 genes,which belong to HSP70.The results of GO enrichment showed that hypothermia mainly affected the biological processes of cells.The enrichment results of Kyoto Encyclopedia of Genes and Genomes（KEGG）showed that the differential genes were mainly enriched in mitogen activated protein kinase（MAPK）and smooth muscle contraction signal pathways.Cell experiments confirmed that HSP70 is a target protein for mild hypothermia to play a protective role,and the use of HSP70 inducers can achieve similar results with hypothermia.Hypothermia plays a role of molecular chaperone by increasing HSP70concentration,protecting mitochondrial function,stabilizing mitochondrial membrane potential,reducing ROS of cells,and ultimately reducing apoptosis.（3）The model of hair follicle culture in vitro was established to considered the damage pathway of taxol to hair follicle and the protection pathway of hypothermia to hair follicle.The results showed that paclitaxel had a mitotic specific effect on the proliferation of human hair follicle stromal keratinocytes,but did not inhibit the proliferation.Paclitaxel promoted the abnormal accumulation of phosphate histone H3（p H3⁺）keratinocytes in hair matrix and outer root sheath rich in stem cells,indicating that mitosis stopped.Taxol promotes the accumulation of irregular and shrunken nuclei.Paclitaxel can significantly increase the number of caspase-3⁺cells.Hypothermia had no significant effect on the expression of Ki-67,but had a significant protective effect on the hair follicles treated with paclitaxel,which prevented the abnormal accumulation of p H3⁺cells in the hair matrix and outer root sheath,and prevented the increase of the number of Caspase-3⁺lytic cells.Paclitaxel has obvious damage to hair follicle epithelial stem cells and induces apoptosis of stem cells,which is the main reason for permanent hair loss caused by chemotherapy.Hypothermia treatment has a certain protective effect on stem cells.The model of chemotherapeutic alopecia and prevention in Kunming（Km）mice was successfully constructed.The back of the model of chemotherapeutic alopecia was treated with cooling,and the hair in the treatment area of the back did not fall off.（4）A double-layer diffusion and dynamic model was established to study the changes of vessel diameter under different temperatures.The results showed that after cold stimulation of skin tissue,the changes of Ca²⁺and phosphorylated myosin increased first,reached the peak value,then oscillated and attenuated,and finally became stable,with the stable value higher than the initial value.The change rule of NO and vessel diameter is decreases first,reaches the valley value,then oscillates and attenuates,and finally tends to be stable,and the stable value is lower than the initial value.And the lower the temperature,the more obvious this trend.When the temperature is 295K,the final change rate of dimensionless diameter of blood vessel is 0.32.NO,Ca²⁺,phosphorylated myosin and tube diameter were successively adjusted,and the time when the tube diameter peak appeared was 28.8 min different from the time when the NO concentration peak appeared.In addition,we also studied the flow characteristics of paclitaxel in capillaries,its transport through capillary walls,and in tissue gaps.Finally,we established a pharmacological model of paclitaxel during scalp cooling,which can predict the concentration of paclitaxel in scalp.The results showed that the concentration of paclitaxel in scalp was significantly lower than that of no scalp cooling.After a period of time,there was no difference in scalp concentration between the two conditions.When the blood perfusion decreased by 5times,the maximum concentration of paclitaxel decreased by 3.5 times,and the average concentration decreased by 1.6 times.The goal of scalp cooling should be that the skin temperature should not be lower than 16℃.Combined with the patient’s cold tolerance,the patient’s chemotherapy dose should not exceed 60-70 mg/m² when the scalp is cooled（16℃）.In conclusion,the key target gene of hypothermia inhibiting taxol induced injury through the cytotoxicity model in vitro was proved in this paper.The mechanism of hypothermia inhibiting taxol induced hair follicle injury was verified.The prediction model of paclitaxel concentration in scalp was established.The results of this paper are helpful to explain the mechanism of inhibiting chemotherapy injury,and provide theoretical and experimental basis for clinical treatment and drug research of subsequent chemotherapy alopecia.