Study On The Improvement Effect And Mechanism Of Steamed Panax Notoginseng On Different Types Of Anemia

[Objectives]Anemia is one of the most common diseases in clinic,which can be induced by a variety of pathophysiological factors.The major symptoms include yellow complexion,asthenia,loss of appetite and so on.According to the Chinese Center for Disease Prevention and Control,the anemia rates of Chinese adult men,women,children,and pregnant women are about 10%,20%,25%,and 35%,respectively.The current treatment for anemia such as supplementing iron,folic acid,and glucocorticoids,is often accompanied by the gastrointestinal irritation,obesity,blood pressure and other side effects.The blood transfusion to treat severe anemia can even lead to hemolysis or heart failure.Therefore,it has been of great concern about how to treat or improve anemia safely and effectively.Panax Notoginsenseng（Burk）F.H.Chen（PN）,as a valuable Chinese herbal medicine,has been known as"natural supplement"in the history of traditional Chinese medicine and folk medicine.There has been a description for PN that“the raw PN materials eliminate and the steamed ones enrich”.The so-called“eliminate”means that the raw PN has the functions of detumescence,relieving pain,promoting blood circulation and removing stasis.The“enrich”means that steamed PN（SPN）has the functions of nourishing blood and qi,and strengthening the body.In recent years,there is an increased attention paid to the blood tonifying effect of SPN.And no related adverse reactions have been reported yet.Although there has been some progress on the pharmacologic research of SPN,most of these studies have only been focused on the therapeutic effect of SPN on single anemia.There is no a systematic and in-depth study on its effect on multiple types of anemia and the specific mechanisms,which not only limits the product development of SPN,but also restricts the choice of clinical drug for anemia.Therefore,three types of anemia models,namely myelosuppressive anemia,renal anemia,and iron deficiency anemia,have been established in the study,to investigate the improvement effect of SPN on these three types of anemia and to explore its underlying action mechanism by using modern pharmacologic,molecular biological and network pharmacology analysis.[Methods]1.PN samples were collected to prepare the steamed form.2.The myelosuppressive anemia model was established by giving cyclophosphamide and acetylphenylhydrazine to the mice.After the model was established,mice were given different doses of SPN and positive control drug for 12 days.The quantities of peripheral blood cells and organs coefficients were determined.The m RNA expression of hematopoietic function-related cytokines（EPO,EPOR,TPO,c-Mpl,GM-CSF and GATA1）in the bone marrow of mice was detected by RT-q PCR.The JAK-STAT signaling pathway was screened based on our previous analysis by network pharmacology.The expression of related proteins（IL-2,STAT1 and SHP2）and cell cycle factors（Bcl-2,Bcl-XL,C-myc and p21）predicted in the pathway was determined by Western blot and RT-q PCR.3.The mice model with renal anemia was established by giving the mice adenine.After the model was established,mice were given different doses of SPN and positive control drug for 30 days.The quantities of peripheral blood cells number and erythrocyte fragility were determined.The renal function indexes（SUN and Scr）and the content of oxygen free radical scavenger（GSH,GSH-Px,SOD）in serum were determined.The pathologic morphology of kidney tissue was determined by HE staining.The m RNA expression of EPOR in BMNCs,EPO,TGF-β₁ and HGF in kidneys of mice was detected by RT-q PCR.The contents of Bcl-2and Bax in kidney were determined.4.The mice model with iron deficiency anemia was established by feeding the mice with a low-iron diet.After the model was established,mice were given different doses of SPN and positive control drug for 30 days.The quantities of peripheral blood cells and organs coefficients were determined.The contents of SI,SF,s TRF,s Tf R and EPO in serum were determined.The m RNA levels of DMT-1,FPN-1 in duodenum and hepcidin in liver were determined by RT-q PCR.[Results]1.For the myelosuppressive anemia model,the levels of red blood cell（RBC）,white blood cell（WBC）,platelet（PLT）and hemoglobin（Hb）in mice treated with high-dose SPN（H-SPN）were significantly higher than that in the model group（P<0.01 or P<0.05）.The level of WBC in mice treated with H-,middle-and low-dose SPN（M-SPN and L-SPN）was significantly increased（P<0.01）.And the spleen coefficient in mice treated with H-and M-dose SPN was significantly decreased（P<0.01 or P<0.05）.The m RNA expression of EPO,EPOR,TPO and c-Mpl in bone marrow nucleated cells of mice with myelosuppressive anemia was significantly increased after the administration of H-SPN（P<0.01）.And the m RNA levels of GM-CSF and GATA1 were significantly increased in the H-,M-and L-SPN groups（P<0.01 or P<0.05）.According to the results of Western blot,there was no significant difference in JAK1 protein expression among different groups（P>0.05）.The p-JAK1 levels in H-and M-dose SPN groups were significantly lower than that of the model group（P<0.01）.The levels of IL-2 protein in mice spleen of H-,M-and L-SPN groups were significantly increased（P<0.01 or P<0.05）.H-SPN significantly increased the expression of STAT1 and SHP2 m RNA（P<0.05）,whereas the M-and L-SPN made no significant effect（P>0.05）.The expression of IL-2 and SHP2 m RNA in spleen of mice with myelosuppressive anemia was significantly increased after treated with H-and M-SPN（P<0.01）.And the expression of STAT1 m RNA was significantly decreased（P<0.01）.The expression of JAK1 m RNA was significantly decreased after treated with H-SPN（P<0.01）.Meanwhile,the levels of Bcl-2and Bcl-XL m RNA in spleen of mice with myelosuppressive anemia were significantly increased after treated with H-and M-SPN（P<0.01）.In addition,the level of Bcl-XL m RNA was also significantly increased after treated with L-SPN（P<0.01）.The levels of c-Myc and p21 m RNA were significantly decreased when given H-and M-SPN（P<0.01 or P<0.05）.2.For the renal anemia model,the contents of RBC,WBC,Hb and HCT in the H-SPN group were significantly higher than those in the model group（P<0.01）.H-SPN significantly increased the levels of WBC and hematocrit（HCT）（P<0.01）,and significantly increased the volume of Hb（P<0.05）.The content of SUN in serum of mice treated with H-and M-SPN was significantly decreased（P<0.01 or P<0.05）.And the content of Scr in serum of mice treated with H-,M-and L-SPN was significantly decreased（P<0.01 or P<0.05）.H-,M-and L-SPN significantly reduced the 50%hemolysis rate of red blood cells（P<0.01 or P<0.05）.The contents of GSH,GSH-Px and SOD in the serum of mice treated with H-and M-SPN were significantly increased（P<0.01 or P<0.05）,while there was no significant difference in the L-SPN group（P>0.05）.The expression of EPO m RNA in renal of mice treated with H-and M-SPN was significantly increased（P<0.01 or P<0.05）.And the expression of EPOR m RNA in bone marrow of mice treated with H-,M-and L-SPN was significantly increased（P<0.01）.H-,M-and L-SPN can significantly improve the pathological condition of kidney tissues in renal-anemia mice.The expression of TGF-β₁ m RNA in the kidney of mice treated with H-and L-SPN was significantly decreased（P<0.01 or P<0.05）.And the expression of HGF m RNA treated with H-SPN was significantly increased（P<0.01）.Meanwhile,the content of Bcl-2 in the kidney of mice treated with H-,M-and L-SPN was significantly increased（P<0.01 or P<0.05）,while H-and M-SPN significantly decreased the content of Bax（P<0.01）.3.For the model of iron deficiency anemia model,the levels of RBC and PLT in mice treated with H-and M-SPN were significantly increased（P<0.01）.The level of HCT in mice treated with H-SPN was significantly increased（P<0.05）.And the level of Hb in mice treated with H-and L-SPN was significantly increased（P<0.01 or P<0.05）.The liver coefficient in mice treated with H-SPN was significantly increased（P<0.05）.And the spleen coefficient in mice with H-and M-SPN was significantly increased（P<0.01 or P<0.05）.The iron content in liver of mice treated with H-,M-and L-SPN was significantly increased（P<0.01 or P<0.05）.And the iron content in spleen of mice treated with H-and M-SPN was significantly increased（P<0.01 or P<0.05）.The content of SI in serum of mice treated with H-and M-SPN was significantly increased（P<0.01）.The content of SF in serum of mice treated with H-,M-and L-SPN was significantly increased（P<0.01 or P<0.05）.The content of TRF in serum of mice treated with H-,M-and L-SPN was significantly decreased（P<0.01 or P<0.05）.And the contents of s Tf R,TIBC and EPO in serum of mice treated with H-and M-SPN were significantly decreased（P<0.01 or P<0.05）.The levels of DMT-1 and FPN-1 m RNA in duodenum of mice treated with H-and M-SPN were significantly decreased（P<0.01）.The expression of DMT-1 m RNA was significantly decreased in L-SPN groups（P<0.05）.And the expression of hepcidin m RNA in liver of mice treated with H-,M-and L-SPN was significantly increased（P<0.01 or P<0.05）.[Conclusion]Based on the blood-nourishing effect of SPN in the theory of traditional Chinese medicine,modern pharmacologic models were used to evaluate the improving effect of SPN on different types of anemia.Combined with our previous prediction result of network pharmacology,the action mechanism of treating anemia by SPN was studied at the level of molecular biology.According to the results,SPN improved anemia by increasing the levels of peripheral blood parameters,protecting metabolism of blood cells and immune organs.regulating the expression of hematopoiesis-related factors,promoting the proliferation and differentiation of hematopoietic stem/progenitor cells,regulating the cycle of damaged cells,and improving the hematopoietic microcirculation.In conclusion,SPN exerts its anti-anemia effect through multiple levels,factors and targets.This study provides clues for the clinic use of drugs alleviating anemia and further guidance for the drug development of SPN.