| ObjectiveAplastic anemia?AA?is a bone marrow failure disease caused by various causes.It is characterized by hypoplasia of bone marrow and decrease of whole blood cells.The main clinical manifestations are anemia,hemorrhage and infection.At present,the etiology of AA is still unclear,which may be related to physical radiation,chemical drugs,viruses and genetic factors.The pathogenesis of AA is complex,mainly involving hematopoietic stem/progenitor cell deficiency,immune abnormality and destruction of hematopoietic microenvironment.The incidence of AA showed a bimodal distribution curve,with one peak between 15 and 25years old and the second peak over 60 years old.There was no significant difference between male and female.Clinical treatment mainly includes hematopoietic stem cell transplantation and immunosuppressive therapy,but both of them have their shortcomings and limitations.Therefore,the search for safer and more effective treatment methods is still a hot spot of current research.Low intensity pulsed ultrasound?LIPUS?is a kind of ultrasound mainly featuring cavitation effect and mechanical effect.Our previous experiments showed that LIPUS treatment significantly promoted the proliferation of bone marrow nucleated cells,increased the number of white blood cells and immunoglobulins in peripheral blood,and decreased the diarrhea rate and mortality rate in chemotherapeutic bone marrow-suppressed animals;LIPUSpromotedthemigrationof hematopoietic cells to peripheral blood by reducing the expression of adhesion molecules on CD34 positive cells;and by reducing the expression of PU.1,it promoted HSCs to granulocyte.Differentiation of lymphocyte.The purpose of this study was to establish an animal model of AA induced by benzene and cyclophosphamide in New Zealand rabbits,and to explore the efficacy and mechanism of LIPUS in treating AA.Methods1.Establishment of AA animal model by combination of benzene and cyclophosphamide:benzene 1.0ml/kg,8 injections,cyclophosphamide15mg/kg,3 injections,which is one dosing cycle,a total of 3 cycles.A New Zealand rabbit AA model with hypoplasia of bone marrow and hematopenia was established.2.16 AA model rabbits were randomly divided into control group and LIPUS group with 8 rabbits in each group.Ultrasound irradiation?0.2W/cm2,0.6 MHz,20 min?was performed on the metaphysis of femur in LIPUS group once a day for 30 days.The control group received false irradiation.The general situation of New Zealand rabbits was observed.Peripheral blood cell count was measured by blood routine test.Bone marrow proliferation was observed by pathological section of bone marrow tissue.Changes of immune-related molecules in peripheral blood were detected by ELISA.3.Cell experiment was divided into four groups:BMSC group:BMSCs were cultured separately;BMSC+Th17 group:BMSCs were inoculated on six-well plate and then added to Th17 cells;BMSC+Th17+LIPUS group:BMSCs were inoculated on six-well plate first,then added to Th17 cells after being covered,and the co-cultured cells were irradiated with LIPUS once after overnight?30mW/cm2,0.6 MHz,20 min?;Th17group:Th17 cells were cultured separately.The four groups of cells were collected at the same time.The levels of IL-6,IL-17A and IL-17F were detected by ELISA,and the expression of STAT3 in Th17 cells was detected by RT-PCR and WB.Results1.Before administration,the WBCs,RBCs,PLTs and HGB counts of the 32 New Zealand rabbits were 15.13±5.12×109/L,8.82±0.58×1012/L,234±107×109/L and 311±20g/L,respectively.After administration,the WBCs,RBCs,PLTs and HGB counts were 5.55±5.55×109/L,7.06±0.54×1012/L,167±35×109/L and 251±14g/L,respectively.WBCs,RBCs,PLTs and HGB counts of model rabbits decreased by 63.3%,20.0%,28.6%and19.3%,respectively,and the differences were statistically significant compared with normal rabbits?P<0.05?.The bone marrow hematopoietic tissue of normal New Zealand rabbit is abundant and the cell proliferation is active.The normal structure of bone marrow tissue was destroyed,hematopoietic tissue decreased,adipose tissue increased,bone marrow granule was empty,and hematopoietic cells decreased.2.The model rabbits showed reduced activity,slow response,reduced food and water intake,and hair loss,which improved after LIPUS irradiation.There was no significant difference in mortality between the LIPUS group?12.5%?and the control group?25%??P>0.05?.At day 151of the experiment,WBCs,RBCs,PLTs and HGB counts in the LIPUS group were 11.53±6.38×109/L,10.12±0.83×1012/L,239±32×109/L and253±11g/L,respectively.In the control group,WBCs,RBCs,PLTs and HGB counts were 9.23±3.70×109/L,8.56±0.60×1012/L,149±32×109/L and 224±13g/L,respectively.There were statistically significant differences in RBCs,PLTs and HGB counts between the two groups?P<0.05?.Compared with the 93rd day of the experiment,there was no significant change in the number of bone marrow nucleated cells in the control group at the 123rd day of the experiment?P>0.05?,while there was a significant increase in the number of bone marrow nucleated cells in the LIPUS group?P<0.05?.Serum FOXP3 levels increased after LIPUS treatment,while IL-17 levels decreased significantly compared with the control group?P<0.05?.After treatment with LIPUS,serum IL-4 and IFN-?levels showed the same trend,with no statistically significant difference?P>0.05?.3.The lowest level of STAT3 protein expression in Th17 cells at 0.5 h after LIPUS irradiation is the point at which the inhibitory effect of LIPUS is most pronounced.WB and PCR showed that BMSCs inhibited the expression of STAT3 gene in Th17 cells,and LIPUS directly or through BMSCs indirectly inhibited the expression of STAT3 gene in Th17 cells.ELISA showed that Th17 cells inhibited IL-6 secretion of BMSCs,and LIPUS inhibited IL-6 secretion of BMSCs directly or indirectly through Th17 cells.Conclusions1.The adjusted administration method can establish the AA animal model of New Zealand rabbit with decreased myeloproliferative and peripheral blood tri-line cells.2.LIPUS can not only increase the number of WBCs,RBCs,PLTs and HGB in peripheral blood,but also promote the proliferation of nucleated cells in bone marrow and improve the fatty state of bone marrow.It can also regulate the level of immune molecules in peripheral blood and correct the abnormality of immune molecules.3.LIPUS can directly or indirectly inhibit the secretion of IL-17 by Th17 cells by reducing the secretion of IL-6 by BMSCs,thereby improving the imbalance of FOXP3/IL-17 ratio in model rabbits. |
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