ObjectiveTo investigate the effect of focal ischemic preconditioning (IPC) on the expression of protein kinase-like endoplasmic reticulum kinase ( PERK ) and glucose-regulated protein 78 (GRP78) mRNA and protein after focal cerebral ischemia/reperfusion (I/R) in rats.MethodsAll 120 male SD rats were randomly divided into three groups: sham-operation group,middle cerebral artery occlusion (MCAO) group and brain ischemia preconditioning (BIP) group.Each group was further divided into 4 subgroups according to 12 h,1,2 and 3 d after I/R.The IPC models were made in order to measure the expression of PERK,GRP78 mRNA and protein by in situ hybridization and Western blot,and the apoptosis rate of neuron by flow cytometry. Results ①The expression of PERK mRNA increased and reached the peak at 12 h,then decreased continuously after 1 d.BIP could decrease its expression.The expression of PERK protein increased at 12 h and reached the peak at 24 h,then decreased continuously after 2 d.BIP could decrease its expression.②The expression both of GRP78 mRNA and its protein all increased and reached the peak at 12 h,then decreased continuously.BIP could increase their expression (mRNA:12 h: 136.70±9.53,F=32.265; 24 h:147.54 ±9.97,F=54.920; 2 d:158.16 ±9.44,F=45.374; 3d: 165.85±10.26,F=16.493,P＜0.05; protein:12 h: 1.319±0.116,F=5.619,P＜0.05; 24 h: 1.226±0.108,F=33.742,P＜0.01; 2 d:1.183 ±0.112,F =46.556,P ＜0.01; 3 d:1.115± 0.098,F =11.730,P＜0.05).③The rate of apoptosis neuron of rats in MCAO increased markedly at 12 h after reperfusion,and reached the peak at 1 d,then decreased continuously.BIP could decrease the rate of apoptosis neuron. Conclusion BIP can protect neurons through inhibiting the expression of PERK and inducing the expression of GRP78 after endoplasmic reticulum stress in rats.

This study was aimed to explore the function of c-Jun N-terminal kinase (JNK) signaling pathway in the induction of brain ischemic tolerance, and observe the function of Shu-Xue Tong-Mai (SXTM) capsule pretreatment. Ischemic preconditioning was performed for 3 min on rats to induce cerebral ischemic tolerance. Rat model of cere-bral ischemia reperfusion (the ischemia pretreatment group, I/R group) was established 24 h later. Western blot was used to detect the protein expression of JNK and phosphorylation of c-Jun N-terminal kinase (P-JNK), comparing to the expression with the sham operation group, I/R group and SXTM capsule group. Tunel method was applied to de-tect the apoptosis of neurons. Relationship between expression of JNK, P-JNK and apoptosis of neurons was also studied. The results showed that compared with the model group, expressions of P-JNK in ischemia preconditioning group and SXTM group were declined significantly (P < 0.05); and the apoptosis of neurons quantity was also de-clined (P< 0.05). It was concluded that ischemia preconditioning can decrease the apoptosis of neurons in cerebral ischemia reperfusion, and improve neurologic function. Its mechanism related to the inhibition of JNK signaling path-way. SXTM capsule pretreatment can protect the cerebral by inhibiting the JNK signaling pathway.

Objective To investigate the role of Calcineurin binding protein 1(Cabin1)in renal tubular epithelial cells(RTECs)injury. Methods The male Sprague-Dawley rats were randomly divided into Sham-oper-ated and 5/6 nephrectomized group. Nephrectomized rats were further divided into two groups ,which were 4 and 8 weeks after operation,including 6 rats in each group. Rats were sacrificed at 4 or 8 weeks after nephrectomy,then control or remnant kidneys were harvested. 2μm sections of kidney tissues were collected and stained with Masson's trichrome and were graded for tubulointerstitial lesion score (TILS). RTECs mitochondrial morphology changes were detected by electron microscope. Western blot was applied to detect Cabin1 protein level in the renal tissue. Results At 8 weeks after the operation,plenty of RTECs fell off from the basement membrane,accompanied with interstitial fibrosis and the infiltration of inflammatory cells. Moreover ,TILS were significantly increased in rats at 8 weeks after operation while compared to sham-operated rats(7.16 ± 0.52 vs. 0.00 ± 0.00,P<0.05). RTECs mi-tochondria begun to swell at 4 weeks after 5/6 nephrectomy,while the disruption of cristae could be found in rats at 8 weeks. Cabin1 protein expression apparently increased in the remnant kidney. Cabin1 protein obviously increased in rats at 8 weeks after the surgery compared to sham-operated rats(0.97 ± 0.09 vs. 0.22 ± 0.07,P<0.05)and rats at 4 weeks after nephrectomy(0.97 ± 0.09 vs. 0.45 ± 0.03,P<0.05). Conclusions Cabin1 is overexpressed during RTECs injury in 5/6 nephrectomized rats. It can be a crucial factor regulating the damage of RTECs.

Background and Objectives: Cancer stem cells (CSCs) with tumorigenic potential are reported as the crucial factors of hepatocellular carcinoma (HCC) recurrence and therapy resistance. Bone mesenchymal stem cells (BMSCs) are documented to play an important role in the protection of hepatocytes. Bie Jia Jian pill (BJJP), a Traditional Chinese Medicine, has been used to treat liver fibrosis and liver cancer. This study aimed to explore the potential role of combined use of BJJP with BMSCs in HCC cell lines. Methods: and Results: Flow cytometry was used to identify BMSCs isolated from BALB/c mice and CSCs enriched from Huh7 cells by measuring CD24, CD133, CD44, CD73, CD105, CD166, CD29, CD14 and CD34. Differentiation potential of BMSCs was also determined. Cell viability and proliferation ability of CSCs were determined by CCK-8 assay and clone formation assay. The expressions of CSCs biomarkers and Wnt/β-catenin signal pathway related proteins were determined by PCR and western blot. TOP-Flash/FOP-Flash luciferase assay was applied to measure the activity of β-catenin/TCF. Compared with untreated CSCs, BJJP or BMSCs treatment alone on CSCs lead to increased miR-140 expression and cell apoptosis, as well as decreased expressions of CD24, CD133, EpCAM and cell viability.Downregualted expressions of Wnt/β-catenin signal pathway related proteins, Wnt3a and β-catenin were found in response to BJJP or BMSCs treatment alone. The combination of BJJP＋BMSCs treatment on CSCs could further enhance the suppressive effect on CSCs. Down-regulation of miR-140 in CSCs partially blocked the effects of BMSCs or BMSCs＋BJJP on the expressions of Wnt3a and β-catenin as well as the cell viability and apoptosis of CSCs.Reversed expression pattern was found in CSCs transfected with miR-140 overexpression. Conclusions Taken together, we demonstrate that BJJP＋BMSCs together could further enhance the suppressive effect on CSCs through regulating miR-140 and suppressing Wnt/β-catenin signal pathway. This study demonstrated the potential of BJJP＋BMSCs in therapeutic treatment of HCC.

Background and Objectives: Cancer stem cells (CSCs) with tumorigenic potential are reported as the crucial factors of hepatocellular carcinoma (HCC) recurrence and therapy resistance. Bone mesenchymal stem cells (BMSCs) are documented to play an important role in the protection of hepatocytes. Bie Jia Jian pill (BJJP), a Traditional Chinese Medicine, has been used to treat liver fibrosis and liver cancer. This study aimed to explore the potential role of combined use of BJJP with BMSCs in HCC cell lines. Methods: and Results: Flow cytometry was used to identify BMSCs isolated from BALB/c mice and CSCs enriched from Huh7 cells by measuring CD24, CD133, CD44, CD73, CD105, CD166, CD29, CD14 and CD34. Differentiation potential of BMSCs was also determined. Cell viability and proliferation ability of CSCs were determined by CCK-8 assay and clone formation assay. The expressions of CSCs biomarkers and Wnt/β-catenin signal pathway related proteins were determined by PCR and western blot. TOP-Flash/FOP-Flash luciferase assay was applied to measure the activity of β-catenin/TCF. Compared with untreated CSCs, BJJP or BMSCs treatment alone on CSCs lead to increased miR-140 expression and cell apoptosis, as well as decreased expressions of CD24, CD133, EpCAM and cell viability.Downregualted expressions of Wnt/β-catenin signal pathway related proteins, Wnt3a and β-catenin were found in response to BJJP or BMSCs treatment alone. The combination of BJJP＋BMSCs treatment on CSCs could further enhance the suppressive effect on CSCs. Down-regulation of miR-140 in CSCs partially blocked the effects of BMSCs or BMSCs＋BJJP on the expressions of Wnt3a and β-catenin as well as the cell viability and apoptosis of CSCs.Reversed expression pattern was found in CSCs transfected with miR-140 overexpression. Conclusions Taken together, we demonstrate that BJJP＋BMSCs together could further enhance the suppressive effect on CSCs through regulating miR-140 and suppressing Wnt/β-catenin signal pathway. This study demonstrated the potential of BJJP＋BMSCs in therapeutic treatment of HCC.

Objective To investigate the protective mechanism of Wenyang Fuyuan Prescription on nerve injury by improving brain iron metabolism in rats with cerebral ischemia-reperfusion injury(CIRI)based on ferroptosis.Methods A total of 72 SD rats were randomly divided into sham-operation group,CIRI model group,Wenyang Fuyuan Prescription group(18.0 g·kg-1,gavage),ferroptosis inducer group(100 mg·kg-1,intraperitoneal injection),Wenyang Fuyuan Prescription(18.0 g·kg-1,gavage)+ ferroptosis inducer group(intraperitoneal injection)and ferroptosis inhibitor group(5 mg·kg-1,intraperitoneal injection),12 rats in each group.All the procedures adopted in the sham group were the same as those in the model group.But nylon thread was inserted into the internal carotid artery at a depth of 9 mm and un-plugged middle cerebral artery.The rest of the groups were used to construct middle cerebral artery occlusion/reperfusion(MCAO/R)model by thread embolism method.Ferroptosis inducer(100 mg·kg-1)and ferroptosis inhibitor(5 mg·kg-1)were administered intraperitoneally to rats according to the grouping 24 hours before modeling.Wenyang Fuyuan Prescription(18.0 g·kg-1)was administered by gavage 2 hours after anesthesia and awakening.All intervention were given once daily for 7 consecutive days.The Longa scoring standard was used to evaluate the neurological deficit on 1,3,and 7 days after MCAO/R surgery,respectively.At the end of the treatment period,brain tissues were taken to observe the morphological changes of rat neurons in each group by hematoxylin eosin staining(HE).The ultrastructural changes of neuron mitochondria in each group were observed by transmission electron microscope.The biochemical kit was used to detect the content of iron ions(Fe2+)and reduced glutathione(GSH)in brain tissue.The protein and mRNA expressions of transferrin receptor 1(TFR1),iron regulatory protein 1(IRP1)and ferroportin(FPN)were detected by real-time quantitative polymerase chain reaction(RT-qPCR)and Western Blot.Results① Compared with sham group,the neurological deficit scores of rats in model group increased at each time point(P＜0.01).HE staining showed neurons were sparse and disordered,the nuclei underwent pyknosis,and vacuoles appeared at the edges.Under electron microscopy,it was observed that the number of neuronal mitochondria decreased,the density of mitochondrial membranes increased,massive numbers of mitochondrial membranes ruptured and dissolved,and mitochondrial cristae disappeared.The content of Fe2+,both mRNA and protein expressions of TFR1 were significantly increased(P＜0.01),while GSH content,as well as expressions of mRNA and protein for IRP1 and FPN were significantly decreased(P＜0.05,P＜0.01).② Compared with the model group,the neurological deficit scores of rats in the Wenyang Fuyuan Prescription group decreased at various time points(P＜0.05).The number of neurons increased,their arrangement was relatively neat,the morphology of the nucleus is complete and clear,the mitochondrial structure of neurons was relatively complete,the mitochondrial membrane was relatively intact,and the mitochondrial cristae were clear.The content of Fe2+,both mRNA and protein expressions of TFR1 were decreased(P＜0.05,P＜0.01),while GSH content,as well as expressions of mRNA and protein for IRP1 and FPN increased(P＜0.05,P＜0.01).③ Compared with the Wenyang Fuyuan Prescription group,the neurological deficit scores of rats in ferroptosis inducer group and the Wenyang Fuyuan Prescription + ferroptosis inducer group increased at all time points(P＜0.05).Distribution of neurons was in disorder,the nucleus shrinked,and vacuoles appeared at the edges.The density of mitochondrial membranes increased,some ruptured and dissolved mitochondrial membranes were found.The number of mitochondria decreased and mitochondrial cristae disappeared.The content of Fe2+,both TFR1 mRNA and protein expression increased(P＜0.05,P＜0.01),while the content of GSH,as well as expressions of mRNA and protein for IRP1 and FPN decreased(P＜0.05,P＜0.01).However,there was no statistically significant difference in all observed indicators between the ferroptosis inhibitor group and the Wenyang Fuyuan Prescription group(P＞0.05).Conclusion Wenyang Fuyuan Prescription can improve the neurological function and pathological damage of CIRI rats.Its mechanism may be related to regulating the expression of IRP1 protein,improving the brain iron metabolism pathway,and inhibiting ferroptosis.

Objective　To investigate the protective mechanism of Rho protein kinase 2 (ROCK2) inhibitor constructed based on adeno-associated virus vector on vascular dementia (VaD) rat hippocampal neurons.Methods　Forty SD rats were randomly divided into sham operation group,model group,ROCK2 interference group (shROCK2),negative control group.The VaD model was prepared by permanent ligation of bilateral common carotid arteries.AAV9-ROCK2-shRNA was injected into the hippocampus of the rat by stereotaxic technique,the negative control group was injected with the same amount of empty vector adeno-associated virus.After 4 weeks,morris water maze was used to test the learning and memory abilities of rats.RT-PCR and Western blot were used to detect the mRNA and protein expression levels of RhoA,ROCK2,MLC,MLCP.Results　Four weeks after operation.The mRNA and protein expressions of the above 4 indicators in hippocampus increased (P<0.01),the synaptic vesicles disappeared,and the structure of hippocampus was chaotic in the model group.Compared with the model and control group,the learning and memory ability of rats in ROCK2 interference group was significantly improved,the mRNA and protein of the above 4 indicators were significantly decreased (P<0.05),and the pre-synaptic vesicles were normal and the organelles were more complete.Conclusion　Rho protein kinase 2 inhibitors can reduce the expression of downstream related proteins by inhibiting RhoA/ROCK2 signaling pathway,then promote neuronal axon regeneration and alleviate cognitive dysfunction of VaD rats.

OBJECTIVE： To investigate the feasibility of multi-payment for drugs for rare diseases and also provide reference for rare disease treatment and the formulation of related policy. METHODS： Taking Gaucher’s disease in Qingdao as an example， the relevant medical insurance policies and drug supply were analyzed； according to the sources of financing， the economic burden of treatment drugs for patients with Gaucher’s disease in Qingdao was estimated. Based on the average total cost of patients with Gaucher’s disease in Qingdao， the drug cost burden of patients with Gaucher’s disease in the whole country was simulated according to the relevant epidemiological survey data， and the problems of medical insurance model for rare diseases in Qingdao were analyzed. RESULTS & CONCLUSIONS： A series of medical insurance policies were formulated in Qingdao. The financing mode of medical insurance includes social funds （from Qingdao Charity Federation， drugs are provided by pharmaceutical manufacturers）， medical insurance funds （used for personal self-payment assistance within the scope of medical insurance co-ordination） and civil assistance （used for assistance outside the scope of medical insurance co-ordination）. Imidase is currently the only approved specific drug for Gaucher’s disease in China. The designated physician， treatment and drug-taken system is adopted in Qingdao. By the end of 2017， the annual drug consumption of 8 patients in Qingdao was 38-170 bottles， totaling 686 bottles. The annual cost of treatment ranged from 786 600 to 3 519 000 yuan， totaling 1 420 200 yuan. The self-paid expenses ranged from 9 800 to 197 400 yuan （the self-paid ratio ranged from 0.46％ to 8.87％）， totaling      661 400 yuan. The reimbursement cost by supplementary medical insurance was 509 800 to 1 789 800 yuan （accounting for 54.59％ to 65.94％）， totaling 8 577 800 yuan. Three patients received civil assistance， the amount of which ranged from 23 100 to 13 000 yuan （accounting for 1.89％ to 4.18％）， totaling 241 400 yuan. According to the relevant epidemiological survey data （the prevalence rate was 0.15 per 100 000）， it is estimated that there are about 2 093 patients with Gaucher’s disease in China. Referring to Qingdao multi-payment model， it is estimated that the total cost of drugs for Gaucher's disease in China is about 3.715 billion yuan， charitable assistance can bear 1.238 billion yuan， while medical insurance expenditure is about 2.255 billion yuan， and individual self-payment is about 222 million yuan. From the point of view of drug cost burden， the multi-payment model in Qingdao has lightened the personal burden of patients’ has achieved significant results. There are problems in Qingdao’s multi-payment model， such as “medical insurance immigration”， low drug accessibility， drug price monopoly， three-level disease prevention needs to be strenghten， etc. The state or provinces and cities can refer to Qingdao model when formulating policies related to rare diseases. Great importance should be paid attention to the existing problems.