LRRK2G2019S mutation induced microglia activation after iron removal by inhibiting autophagy associated proteins / 中华神经医学杂志

Objective:To investigate the effect of LRRK2G2019S mutation on activation of microglia after iron deprivation and its mechanism.Methods:(1) Microglia were differentiated from human induced pluripotent stem cells (IPSC) with the help of hematopoietic progenitor cells (HPC) and identified by immunofluorescent staining, and α-synuclein (α-syn) A53T mutant protein was obtained by protein purification technology. (2) Microglia were divided into control group, α-syn group, α-syn+ deferoxamine (DFO) group; phosphate buffer solution (PBS), 1 μmol/L purified α-syn A53T mutant protein, 1 μmol/L purified α-syn A53T mutant protein+30 mmol/L DFO were given respectively for 24 h. Fe 2+ concentration was detected by colorimetry, Rab35 protein expression was detected by Western blotting, intracellular reactive oxygen species (ROS) level was detected by flow cytometry, and interleukin-6 ( IL-6), tumor necrosis factor-α ( TNF-α) and transforming growth factor-β ( TGF-β) mRNA expressions were detected by real time-PCR (RT-PCR); microglia culture supernatant (MCS) in the 3 groups were transfered to SH-SY5Y cells, and SH-SY5Y cell apoptosis was detected by flow cytometry. (3) Bidirectional DNA sequencing was used to detect leucine rich repeat kinase 2 ( LRRK2) gene mutations in microglia treated with 1 μmol/L purified α-syn A53T mutant protein. Microglia were divided into control group, α-syn group and α-syn+GSK3357679A group, and treated with corresponding drugs for 24 h, respectively (LRRK2 inhibitor GSK3357679A concentration: 10 nmol/L), and LRRK2 protein expression was detected by Western blotting; microglia were divided into control group, α-syn group, α-syn+GSK3357679A, and α-syn+GSK3357679A+DFO group, and treated with corresponding drugs for 24 h, Rab35 protein expression was detected by Western blotting, intracellular ROS level was detected by flow cytometry, and IL-6, TNF-α and TGF-β mRNA expressions were detected by RT-PCR. (4) Microglia were divided into control group, α-syn group, α-syn+rapamycin (RAPA) group, and treated with corresponding drugs for 24 h (concentration of autophagy inducer RAPA: 50 nmol/L); protein expressions of Rab35, P62 and microtubule-associated protein light chain 3 II (LC3II) were detected by Western blotting; intracellular ROS level was detected by flow cytometry, and IL-6, TNF-α and TGF-β mRNA expressions were detected by RT-PCR. (5) Microglia were divided into control group, α-syn group, and α-syn+Rab35 group, and treated with corresponding drugs for 24 h (concentration of Rab35 overexpressed plasmids: 1 μg/mL); Rab35, P62, and LC3II protein expressions were detected by Western blotting; ROS level was detected by flow cytometry, and IL-6, TNF-α and TGF-β mRNA expressions were detected by RT-PCR. Results:(1) Immunofluorescent staining showed negative neuronal nuclei (NeuN) expression and positive ionized calcium-binding adapter molecule 1 (Iba1) expression in microglia, and high LRRK2 expression; PcDNA3.1-SNCA-A53T expression plasmid was constructed and α-syn A53T mutant protein was purified. (2) The Fe 2+ concentration in α-syn group was significantly higher than that in control group, and the Fe 2+ concentration in α-syn+DFO group was significantly lower than that in α-syn group ( P<0.05); the Rab35 protein and TGF-β mRNA expressions in control group, α-syn group and α-syn+DFO group were decreased successively, while the IL-6 and TNF-α mRNA expressions were increased successively, with significant differences ( P<0.05); ROS level and SH-SY5Y cell apoptosis rate in control group, α-syn group, α-syn+DFO group were increased successively. (3) Bidirectional DNA sequencing showed that the LRRK2G2019S mutation in microglia was the most obvious after α-syn A53T mutant protein stimulation; compared with the control group, the α-syn group had significantly increased LRRK2 protein expression, while the α-syn+GSK3357679A group had significantly decreased LRRK2 protein expression compared with α-syn group ( P<0.05); compared with the control group, the α-syn group had significantly decreased Rab35 protein and TGF-β mRNA expressions, and statistically increased IL-6 and TNF-α mRNA expressions ( P<0.05); compared with α-syn group, the α-syn+GSK3357679A group had significantly increased Rab35 protein and TGF-β mRNA expressions, and statistically decreased IL-6 and TNF-α mRNA expressions ( P<0.05); compared with α-syn+GSK3357679A group, α-syn+GSK3357679A+DFO group had significantly increased IL-6 and TNF-α mRNA expressions, and significantly decreased Rab35 protein and TGF-β mRNA expressions ( P<0.05). The α-syn group had higher ROS level than the control group, the α-syn+GSK3357679A group had lower ROS level than the α-syn group, and the α-syn+GSK3357679A+DFO group had higher ROS level than the α-syn+GSK3357679A group. (4) Compared with the control group, the α-syn group had significantly decreased Rab35 and LC3II protein, and TGF-β mRNA expressions, and significantly increased P62 protein, IL-6 and TNF-α mRNA expressions ( P<0.05); compared with α-syn group, the α-syn+RAPA group had significantly increased Rab35 and LC3II protein, and TGF-β mRNA expressions, and significantly decreased P62 protein, and IL-6 and TNF-α mRNA expressions ( P<0.05); the α-syn group had higher ROS level than the control group and α-syn+RAPA group. (5) Compared with the control group, the α-syn group had significantly decreased Rab35 and LC3II protein, and TGF-β mRNA expressions, and statistically increased P62 protein, and IL-6 and TNF-α mRNA expressions ( P<0.05); compared with the α-syn group, the α-syn+Rab35 group had significantly increased Rab35 and LC3II protein, and TGF-β mRNA expressions, and significantly decreased P62 protein, and IL-6 and TNF-α mRNA expressions ( P<0.05). The α-syn group had higher ROS level than the control group and α-syn+Rab35 group. Conclusion:LRRK2G2019S can induce neuroinflammation by inhibiting Rab35-related autophagy under iron deprivation, and Rab35 is expected to be a key factor in intervening neuroinflammation.

Influencing factors of iron metabolism assessment in patients with myelodysplastic syndrome: A retrospective study / 中华血液学杂志

Objective: To analyze the influencing factors of iron metabolism assessment in patients with myelodysplastic syndrome. Methods: MRI and/or DECT were used to detect liver and cardiac iron content in 181 patients with MDS, among whom, 41 received regular iron chelation therapy during two examinations. The adjusted ferritin (ASF) , erythropoietin (EPO) , cardiac function, liver transaminase, hepatitis antibody, and peripheral blood T cell polarization were detected and the results of myelofibrosis, splenomegaly, and cyclosporine were collected and comparative analyzed in patients. Results: We observed a positive correlation between liver iron concentration and ASF both in the MRI group and DECT groups (r=0.512 and 0.606, respectively, P<0.001) , only a weak correlation between the heart iron concentration and ASF in the MRI group (r=0.303, P<0.001) , and no significant correlation between cardiac iron concentration and ASF in the DECT group (r=0.231, P=0.053) . Moreover, transfusion dependence in liver and cardiac [MRI group was significantly associated with the concentration of iron in: LIC: (28.370±10.706) mg/g vs (7.593±3.508) mg/g, t=24.30, P<0.001; MIC: 1.81 vs 0.95, z=2.625, P<0.05; DECT group: liver VIC: (4.269±1.258) g/L vs (1.078±0.383) g/L, t=23.14, P<0.001: cardiac VIC: 1.69 vs 0.68, z=3.142, P<0.05]. The concentration of EPO in the severe iron overload group was significantly higher than that in the mild to moderate iron overload group and normal group (P<0.001) . Compared to the low-risk MDS group, the liver iron concentration in patients with MDS with cyclic sideroblasts (MDS-RS) was significantly elevated [DECT group: 3.80 (1.97, 5.51) g/L vs 1.66 (0.67, 2.94) g/L, P=0.004; MRI group: 13.7 (8.1,29.1) mg/g vs 11.6 (7.1,21.1) mg/g, P=0.032]. Factors including age, bone marrow fibrosis, splenomegaly, T cell polarization, use of cyclosporine A, liver aminotransferase, and hepatitis antibody positive had no obvious effect on iron metabolism. Conclusion: There was a positive correlation between liver iron concentration and ASF in patients with MDS, whereas there was no significant correlation between cardiac iron concentration and ASF. Iron metabolism was affected by transfusion dependence, EPO concentration, and RS.

Naringin is a promising natural compound for therapy of iron-overload disorders

Abstract Naringin has been shown to exhibit satisfying iron chelation capacity. Considering the side effects of routinely-used iron chelator (desferrioxamine, DFO), we decided to evaluate the iron chelation potency of naringin to discover whether or not it can be a promising natural substitute for treatment of excessive iron-related diseases. 35 mice were classified into five groups of 7 and subjected to iron dextran administration to induce the iron-overload condition. Iron-overloaded mice were then treated with normal saline (as control), naringin or DFO Morphology changes, and iron deposition in liver tissues were studied using H&E and Perl's staining. The results revealed that naringin is more potent than DFO in removing excessive iron ions deposited in liver tissues, indicating that naringin is a promising natural compound for therapy of iron overload disorders

Beta Thalassemia Major with Diabetes Mellitus: A Case Report

Thalassemia occurs due to defects in normal hemoglobin production. Globally it is most common inherited anaemia. Diabetes is a complication of b-thalassemia major. We report a case of Diabetes mellitus in a known case of beta thalassemia major. Patient had undergone splenectomy 1 year back. Patient is taking chelating agent Defasirox 1000mg orally once a day in the morning. Family history reveals, born through third degree consanguineous marriage. The patient was then subjected for laboratory examination reveals BSLwas 490, urine ketone 2+, urine sugar 3+, ABG was normal, HbA1c was 13 & 3 month old report of serum ferritin 1200 ng/dl. TFTand GH studies normal. Multidisciplinary management was instituted. Blood sugar level got controlled over subcutaneous insulin. Patient may have landed in Diabetic ketoacidosis but was promptly diagnosed & treated. This case is presented for its rarity. Due to increase in life expectancy of patient with thalassemia major, patient will expose more years of hyperglycemia and diabetes. Sustaining metabolic control and controlling cardiovascular risk factors helps to prevent future complications.

Neuroprotective effects of kukoamine A on 6-OHDA-induced Parkinson's model through apoptosis and iron accumulation inhibition / 中草药·英文版

Objective: Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in substantia nigra (SN). Our previous study demonstrated kukoamine A (KuA) to exhibit strong neuroprotective effects through antioxidative stress, and autophagy in MPTP/MPP

Hearing Loss in Thalassemic Children on Chelation Therapy

Background: Thalassemia is a common genetic hematological disorder worldwide. It is also common in North India includingJammu region. These patients need lifelong repeated blood transfusions and iron chelation therapy for their survival. Chelationtherapy is known to be associated with various complications including sensorineural hearing loss (SNHL). Till now, no dataare available regarding SNHL in pediatric thalassemia major patients in Jammu region. Hence, we planned a study to assessthe prevalence of hearing loss in children with thalassemia major in the age group of 10–20 years.Methods: All the children with beta-thalassemia major in the age group of 10–20 years registered with Thalassemia Day CareCenter, Department of Pediatrics, SMGS Hospital, Government Medical College, Jammu, were enrolled in this cross-sectionalstudy. Hearing was assessed by pure tone audiometry. Clinical and demographic data of these patients were recorded onpretested pro forma and analyzed.Results: A total number of 34 children with thalassemia in the age group of 10–20 years were enrolled in this study whichcomprised 18 males and 16 females. Out of these 34 patients, 5 (14.7%) were found to have SNHL and 1 (2.9%) had conductivehearing loss. Four of the five patients in SNHL group had low- as well as high-frequency mild hearing loss (25–40 db) whileone patient had high-frequency mild hearing loss at 4000 HZ. Four out of these five patients had unilateral hearing loss on theleft side while one had bilateral SNHL. Two out of five patients in the SNHL group were taking chelation therapy in the form ofcombination of deferiprone and deferasirox at the dose of 75–100 mg/kg/day and 30–40 mg/kg/day, respectively, for more than5 years. The other three patients were taking only deferasirox at the dose of 30–40 mg/kg/day for more than 5 years.Conclusions: Regular blood transfusions and chelation therapy are essential for long-term survival of thalassemia majorpatients but are also associated with complications like SNHL.

Knowledge, attitude and practice in parents of chronically transfused thalassemic patients regarding thalassemia in thalassemia day care unit in government medical college, Amritsar, Punjab, India

Background: Thalassemia is a chronic debilitating disease that affects nearly 200 million people worldwide. A caregiver who has good knowledge regarding the disease can not only provide a better quality of care to his/her ward but also may spread knowledge in the society in which he/she lives which helps immensely in raising community awareness related to the disease.Methods: 50 caregivers (either mother or father) of chronically transfused thalassemic children were questioned regarding their knowledge, attitude and practice towards this disease, using a self-constructed questionnaire.Results: It was found that despite adequate knowledge regarding every aspect of this disease, there is a lack of attitude of practice towards prevention of this disease in their subsequent child or in their near and dear ones.Conclusions: There is a need to promote an attitude of practice in prevention of the birth of thalassemic children as mere knowledge regarding the disease is not enough in the present scenario where the disease burden is increasing as is the financial and emotional burden on the families. Role of Government sponsorship, a social worker and a child psychologist in the Thalassemia day care unit cannot be minimized.

Impacts of transfusion in β-thalassemia major patients receiving multiple blood transfusions

Background: Beta-thalassemia major is one of major public health problems in India. Thalassemia major is a transfusion-dependent severe anemia and these children experience various problems if the transfusion is inadequate but at the same time repeated blood transfusions are associated with hazards like iron overload.Methods: This study was conducted at thalassemia ward of S.M.S Hospital and J.K.LON paediatrics Hospital, Jaipur from April 2012 to Nov. 2013. 145 thalassemia patients on transfusion therapy attending outdoor or being admitted were assessed after obtaining due permission from the authorities and consent from the guardian or parents of patients.Results: Present study comprised 145 beta-thalassemia major patients with 51 (35%) females and 94 (65%) males in which youngest patient is 3 years old and the oldest 33 years. 104 (72%) were Hindus and 41 (28%) were Muslims. Consanguinity was found in a significant proportion 16% (23) of the parents of the patients. 137 (94%) patients had serum ferritin ³1000ng/ml. Out 145 thalassemic patients, 108 (74.5%) have total serum bilirubin >1(mg/dl), 103 (71%) have SGPT level >35IU, 103 (71%) have SGOT level >40IU), 38 (26.2%) have serum alkaline phosphatase level >390 IU, 35 (24.1%) have serum creatinine level >1.6mg/dl, 42 (29%) have serum urea level >45mg/dl, 28 (19.3%) have serum uric acid level >6mg/dl.Conclusions: It is suggested to revise and devise suitable transfusion regime so that a balance between adequate transfusion and minimum side effects of multiple transfusions is maintained. Systemic effects of multiple transfusions should be rigorously and meticulously studied.

Evaluation Of Free Radical Scavenging Ability And Antiradical Activities Of Ximenia Caffra Fruit Extracts At Different Ripening Stages

Objective: To evaluate the free radical scavenging ability and antiradical activities of Ximenia caffra fruit extracts in their different ripening stages. Methods: Using standard procedures, Ximenia caffra fruit extracts were determined for ferrous ions chelating ability, nitric oxide and hydroxyl radical scavenging ability. Antiradical activities were assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH) in which the fruit extracts were evaluated for the number of antiradical units (AU515), the number of antiradical units per 1 mg of extracts (EAU515) and the total number of antiradical units per 1g of raw fruits (TAU515). Results: The fruit extracts exhibited significant higher ferrous ion chelating and free radical scavenging capacity compared to synthetic antioxidants (standard). Fruit extracts in early ripening stage (ERS) exhibited stronger ion chelation, nitric oxide and hydroxyl radical scavenging ability with low effective fruit extract concentration required to reduce free radicals by 50% (EC50) that were 14, 25 and 30µg/ml respectively than the late ripening stage (LRS) extracts. The fruit extracts also showed high AU515, EAU515 and TAU515. The values of AU515 ranged from 0.93 to 0.95, while EAU515 from 78.30 to 79.34 and TAU515 ranged from 19762.46 to 23821.23 of the extracts in LRS and ERS respectively. Conclusion: Based on these observations Ximenia caffra is potentially beneficial to human health due to its strong ability to scavenge free radicals. Its utilization can potentially reduce the risk of degenerative diseases to human beings.

Study of iron overload assessment by T2* magnetic resonance imaging in patients with myelodysplastic syndromes / 中华血液学杂志

Objectives: To analyze the cardiac T2* value, liver iron concentration (LIC) , and related laboratory parameters in myelodysplastic syndrome (MDS) with iron overload and evaluate the changes of organ functions after iron chelation therapy. To explore the value of magnetic resonance imaging (MRI) T2* in making early diagnosis and assessing organs iron overload. Methods: Retrospective investigation was used to observe the cardiac T2* value, LIC, iron metabolism parameters and related laboratory parameters of 85 MDS patients from Nov 2014 to Jan 2018. Among them, 7 MDS patients with Low/Int-1 have received iron chelation therapy for 6 months during two MRI examinations. The above parameters were collected before and after iron chelation therapy for comparison. Results: Correlations were found between heart T2* value and age (rs=-0.290, P=0.007) and left ventricular ejection fraction (LVEF) (rs=0.265, P=0.009) . There was a significant negative correlation between heart T2* value and blood transfusion units (rs=-0.701, P<0.001) . There was a significant positive correlation between LIC and serum ferritin (SF) (rs=0.577, P<0.001) . There was also a correlation between LIC and ALT (rs=0.268, P=0.014) and blood transfusion units (rs=0.244, P=0.034) . There was no correlation between heart T2* and pro-BNP, SF (all P>0.05) , and no correlation between LIC and age (P>0.05) . The increase of heart T2* between the normal and abnormal groups was statistically significant (P=0.005) , but the iron overload ratio of the heart T2*<20 ms was not significant between the two groups. There was statistical significance in the proportion of severe liver iron overload (LIC>15 mg/g DW) (P=0.045) . After iron chelation therapy, the values of SF, transferrin saturation, ALT, AST, pro-BNP and LIC of 7 patients were decreased compared with values before iron chelation therapy, and the peripheral blood cell level was increased. However, the changes of LVEF and T2* values after iron chelation were not obvious. Conclusion: MRI T2* may be a predictor of iron overload in patients with MDS in early stage, and may be more valuable compare with LVEF, SF and other laboratory indicators. The safety and repeatability of MRI cardiac T2* examination are recognized, and it can be used as an ideal detection for patients with iron overload.