The Molecular Mechanism Of Regeneration Of Jinnangshengbai On Bone Marrow Suppression After Radiotherapy And Chemotherapy

Objective: Global cancer incidence and mortality rates of patients showan increasing trend, surgery, radiotherapy and chemotherapy are still threemajor means for the clinical treatment of cancer. Most advanced cancerpatients and non-solid tumor patients just only choose radiotherapy andchemotherapy. Bone marrow suppression is one of the most common toxicity,the patient’s leukocytes are reduced mainly, even the patient’s pancytopenia,which leading to a serious impact on outcomes. Therefore, in the clinicalcancer therapy field, a focus problem has been raised on how to effectivelyenhance the regeneration of bone marrow suppression.There are two common species of medicine to increase leukocytes inclinic. One is the western medicine, which includes granulocyte colonystimulating factor (G-CSF) mainly. The other is the traditional Chinesemedicine, which includes Diyushengbai tablets, Compound Zaofan pills,Shengbai compost granules and so on. Their application in clinic has beenlimited due to their defects.Jinnangshengbai was conceived by Professor Qi Jinsheng based onexperience of many years. The total efficiency of Jinnangshengbai was up to94%, being a potential novel drug to tolerate radiotherapy and chemotherapytreatments.Hematopoietic stem cells (HSC) is mainly responsible for cellregeneration after bone marrow suppression. With continuous self-renewal,HSC not only maintains a constant number by itself, but meets the demandthrough differentiation into peripheral blood progenitor cell lines. Recentstudies have found that some intrinsic factors regulated HSC self-renewalcapacity strongly, such as the classic WNT signaling pathway, the NOTCH signaling pathway, and the homeobox (HOX) transcription factor family couldregulate HSC self-renewal capacity. It’s considered that there werecross-talking and coordination between NOTCH and WNT signaling pathways.The high level activation of WNT signaling pathway is an important conditionfor hematopoietic progenitor cell proliferation, and NOTCH pathway isnecessary to maintain the undifferentiation and quiescence of hematopoieticprogenitor cell. HOX gene is one of the conserved homeobox gene families.HOX gene regulates proliferation and differentiation in normal hematopoiesis,but its abnormal expression is closely related to leukemia. In addition, anumber of small molecules, such as prostaglandin E2(PGE2), play importantregulatory roles in regulating HSC self-renewal. PGE2may interact with WNTsignaling pathway to promote HSC self-renewal through the activation ofWNT signaling pathway.In this study, the number of leukocytes in irradiated mice was reduced,while increased significantly after treated by Jinnangshengbai. Some genesregulating hematopoietic signaling pathways, such as β-Catenin, NOTCH1,JAGGED1, HOXB4and COX2were detected by PCR and Western blotting,to explore the the Leukopenia model mice drinking JinnangshengbaiPeripheral leukocytosis mechanism. Celecoxib, PGE2synthase COX2specificinhibitor, was added to treat irradiated mice. In order to seekJinnangshengbai’s mechanism in promoting mice hematopoietic recoveryprocess, Celecoxib was used to identify whether PGE2playing a major role toWNT signaling pathway.To further explore effective ingredients or the monomer component ofJinnangshengbai, some active polysaccharide was extracted and purifiedgradually, which is important to elucidate the underlying mechanism and theexploration of Jinnangshengbai.Method:1Animal grouping and drawn Diyushengbai 140healthy male mice (20±5g) were randomly divided into five groups:Diyushen group, Jinnangshengbai group, Celecoxib-Jinnangshengbai group,Pathological control group and Normal control group. Normal control groupwas20mice, and the remaining four groups were30mice. A total of120micefrom the first four groups were intraperitoneal anesthetized by1%sodiumpentobarbital, the linear accelerator X-ray radiated mouse hindlimb bilateralfemur with the distance of100cm, total dose of4Gy. The number of leucocytewas detected one day later, and we succeeded in making model if the decreaseof leucocyte was to5×109.L-1. After the success of modeling, each of thegroups selected20mice. Mice of above five groups had the normal diet.Pathological control group and Normal control group were free to water. Thedosages of the former three groups (Diyushengbai tablets for Diyushengbaigroup, Jinnangshengbai granules for Jinnangshengbai group, andJinnangshengbai granules plus celecoxib capsules for Celecoxib-Jinnangshengbai group) were calculated according to the equivalent doseaccording to human and mice unit weight proportionality, dissolved in waterdemand to the mice daily physiological, and dubbed the correspondingconcentration of the liquid for mice drinking, and then the mice wereadministered continuously for eight days.2Detection of peripheral blood cells of mice and PGE2value in blood serumWithin canthus blood analysis,20μl anticoagulated blood were collectedfeeding at the fourth and eighth day at room temperature20~25℃,responsibility. Peripheral blood cells were detected by the blood cell analysisinstrument. At the same time,200μl anticoagulated blood were collectedfrom each group20mice at the eighth day within canthus blood analysis, wecollected blood serum after natural coagulation at room temperature anddetected PGE2values by ELISA.3Detection of the transcriptional level of COX2, β-Catenin, NOTCH1,JAGGED1, HOXB4contained in MMNCMice were sacrificed by cervical dislocation at the experimental ninth day,we abstracted bone marrow mononuclear cells (MMNC) by using lymphocyte-separating medium, the cells were collected for the extraction oftotal RNA and protein for molecular biology detection like PCR and Westernblotting. We extracted general RNA of MMNC by Trizon with β-actin internalcontrol, the mRNA expression of COX2, β-Catenin, NOTCH1, JAGGDE1,HOXB4was detected by PCR.4The protein content of COX2, β-Catenin, JAGGED1, HOXB4of MMNCWe detected the protein level by Lowry. The protein content of COX2,β-Catenin, NOTCH1, JAGGED1, HOXB4of bone marrow were measured byWestern blotting analysis.Jinnangshengbai was further separated and purified, and the obtainedcomponents were administrated to the mouse model to evaluate theireffectiveness and determine the active position. The active ingredients weredetected by the diode array detector and evaporative light detector to identifythe active component through qualitative analysis.Results:1The general performance of mice, peripheral blood leukocytes and micestate affected by Jinnangshengbai granulesAt the fourth day after treatment, the numbers of peripheral bloodleukocytes (PBLs) in Diyushengbai group (6.8±2.84) and Jinnangshengbaigroup (5.47±1.79) was significantly higher (P<0.05) compared withPathological control group (4.82±2.11). Mice in Jinnangshengbai group hadstronger acytivity and much glossier smooth coat than those of Diyushengbaigroup. There was no significant difference of the white blood cell countbetween Celecoxib-Jinnangshengbai group (3.85±0.79) and Pathologicalcontrol group (4.82±2.11). The general state of mice in Celecoxib-Jinnangshengbai group (3.85±0.79) and Pathological control group were worstof all, and the mice occured a series of appearances such as less activity,listlessness, eyes closed, pale tail and ear in color, coarse hair disorder, fluffyand matt, and their weight decreased with the decrease of food consumption.At the eighth day after treatment, the numbers of PBLs in Diyushengbai group(8.0±3.01) and Jinnangshengbai group (7.99±2.75) was significantly difference (P<0.05) compared with Pathological control group (4.74±2.36).There was no significant difference of white blood cell count betweenCelecoxib-Jinnangshengbai group (3.77±1.74) and Pathological control group(4.74±2.36).2Detection of the concentration of PGE2in mice serumThe concentration of PGE2in mice serum was measured by ELISA, andthe results were that the PGE2concentrations in Jinnangshengbai group andDiyushengbai group were significantly higher (P<0.05) compared withPathological control group, while that of Celecoxib-Jinnangshengbai grouphad no significant difference compared with Pathological control group.3The changes of the transcriptional level of COX2, β-Catenin, NOTCH1,JAGGED1, HOXB4in MMNCThe mRNA expression of COX2in Diyushengbai group(0.1194±0.00795) and Jinnangshengbai group (0.0953±0.01274) weresignificantly higher compared with Pathological control group(0.0377±0.00564). The mRNA expression of Jagged1in Jinnangshengbaigroup (0.2628±0.01855) was significantly higher compared with Pathologicalcontrol group (0.0429±0.00704). There was no significant difference of theβ-Catenin mRNA expression between Jinnangshengbai group(0.3935±0.12128) and Pathological control group (0.423±0.0186). And therewas also no significant difference of the HOXB4mRNA expression betweenJinnangshengbai group (0.0795±0.01407) and Normal control group(0.0757±0.0135).4The protein content of COX2, β-Catenin, JAGGED1, HOXB4whichcontained in MMNCThe protein expression of COX2in Diyushengbai group (0.7963±0.099)and Jinnangshengbai group (0.7074±0.0842) were significantly different (P<0.01) compared with Pathological control group (0.0377±0.00564). Theprotein expression of β-Catenin in Diyushengbai group (0.8366±0.1143) andJinnangshengbai group (0.4268±0.0352) were significantly different (P<0.01)compared with Pathological control group (0.0979±0.0151). The protein expression of JAGGED1in Jinnangshengbai group (0.4901±0.0308) andCelecoxib-Jinnangshengbai group were significantly different (P<0.01)compared with Pathological control group (0.1017±0.0078). The HOXB4protein expression in Jinnangshengbai group (0.203±0.013) was no significantdifference compared with Normal control group (0.2249±0.0193).Method:1Extracting the effective constituent of JinnangshengbaiTraditional Chinese medicine was decocted and then extracted throughorganic solvent (OS). The liquid poured and retained in the dregs after soakingwith moderate95%alcohol for24hours. Then the dregs were soaked foranother10hours using the equal amount95%alcohol. The two liquid isethanol phase. the ethanol phase was evaporated and concentrated at60~65℃for two hours, and dried by water bath and then put it into the oven at80℃todry into powder. Organic solvent extraction: first step, ligarine whose boilingrange is60~90℃at the normal atmospheric pressure was used to extractmedicine juice twice. The ligarine phase was evaporated and concentratedwith the temperature40to45℃for two hours, and dried by water bath andthen put it into the oven at80℃to dry into powder. Second step, chloroformwhose boiling point is61℃was used to extract the water phase twice. Thechloroform phase was evaporated and concentrated with the temperature40to45℃for two hours, and dried by water bath and then put it into the oven at80℃to dry into powder. Third step, ethyl acetate was used to extract the waterphase twice, the ethyl acetate phase was evaporated and concentrated with thetemperature40to45℃for two hours, and dried by water bath and then put itinto the oven at80℃to dry into powder. Fourth step, n-butanol was used toextract the water phase twice, the n-butanol phase was evaporated andconcentrated with the temperature70to78℃for two hours, and dried bywater bath and then put it into the oven at80℃to dry into powder. The waterphase was dried by water bath and then put it into the oven at80℃to dry into powder.2Experimental animals and groups230male healthy mice (20±5g) were randomly divided into eight groups:petroleum ether group, chloroform group, ethyl acetate group, n-butyl alcoholgroup, the aqueous phase group, pathological control group and normalcontrol group. Normal control group was20mice, and the remaining sevengroups were30mice. A total of210mice from the first six groups wereintraperitoneal anesthetized by1%sodium pentobarbital, the linear acceleratorirradiation source radiated mouse hindlimb bilateral femur with the distance of100cm, total dose4Gy. The number of leucocyte was detected one day later,and we succeeded in making model if the decrease of leucocyte was to5×109.L-1. After the success of modeling, each of the first seven groupsselected20mice. Mice of above eight groups had normal diet, pathologicalcontrol group and normal control group was free to water, the dosages of theformer five groups(petroleum ether group, chloroform group, ethyl acetategroup, N-butyl alcohol group, the aqueous phase group) were calculatedaccording to the equivalent dose according to human and mice unit weightproportionality, dissolved in water demand to the mice daily physiological,and dubbed the corresponding concentration of the liquid for mice drinking,and then the mice were administered continuously for eight days.3Detection of peripheral blood cells of miceWithin canthus blood analysis,20μl anticoagulated blood were collectedfrom20mice of each group at the eighth day after administration, the changeof peripheral blood cells was detected by automatic blood cell counter.4Detection of the separated substances by liquid chromatography detectorThe presence of ultraviolet absorption of compound was detected in threephase substances which contained the chloroform phase, the ethyl acetatephase, and the aqueous phase with diode array detector, and the presence ofultraviolet absorption of organic substances was detected with evaporativelight detector. Results:1The general performance of mice, PBLs and mice state affected by separatedingredients of Jinnangshengbai Prescription.At the eighth day after treatment, the numbers of PBLs in chloroformgroup (11.97±4.65), ethyl acetate group (12.44±5.13) and the aqueous phasegroup (17.39±6.38) was significantly higher (P<0.05) compared withpathological control group (4.27±2.12). Mice in Tips liter white group Mice inchloroform group, ethyl acetate group and the aqueous phase group hadstronger activity and much glossier smooth coat than those of pathologicalcontrol group. The white blood cell count in ligarine group (6.61±1.96),n-butanol group (5.29±1.79) and alcohol group (7.2±2.78) were no significantdifference compared to pathological control group (4.27±2.12). The mice inthese groups occured a series of appearances such as less activity, listlessness,eye closed, pale tail and ear in color, coarse hair disorder, fluffy and matt, andtheir weight decreased with the decrease of food consumption.2The pharmaceutical separation qualitative analysis of the drugThe data from diode array detector with the wavelength of200to400showed that, there was no significant difference among chloroform group,ethyl acetate group and the water phase group, whose wave types were similar,and there was no UV-absorbing material. Evaporative light detector515X2(alteeh) detected of non-volatile molecules, and determined there was noUV-absorbing substances (polysaccharides without UV absorption). Alcoholprecipitation was macromolecules with the weight of more than5000, and theprecipitation was not much, There might be small molecular polysaccharideswith the weight of5000or less.Conclusion:1Jinnangshengbai could increase the count of peripheral bloodleukocytes in irradiated mice significantly.2The mechanism of Jinnangshengbai promoting leukocytosis was thathigh concentration PGE2was produced after activated the expression of COX2,which then enhanced β-Catenin content to activate the WNT signalingpathway, and promoted HSC proliferation finally. 3Jinnangshengbai could increase Jagged-1mRNA and protein levels. Itsuggested that Jinnangshengbai might activate the NOTCH signaling pathway,and regulate HSC proliferation.4The pharmaceutical separation qualitative analysis of Jinnangshengbaishowed that the active pharmaceutical ingredients were polysaccharide ofmolecular weight less than5,000.