[ABSTRACT]AIM:Toinvestigatecellapoptosisindiabeticfootulcersandtheeffectofadvancedglycosylation end products (AGEs) on apoptosis in human fibroblast cells.METHODS: Diabetic foot patients (n=18) and 18 age-matched non-diabetic controls were recruited .The clinical and biochemical features were compared by statistics methods . Skin biopsies were obtained from foot .Cleaved caspase-3 was measured by immunohistochemistry using the technique of streptavidin-biotin complex ( SABC ) staining.Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling ( TUNEL) technique was used to detect apoptosis of the skin tissues .Human primary foreskin fibroblasts were isolated and cultured in the presence of 5.6 mmo/L glucose, 25 mmo/L glucose, fluctuant glucose ( changing the glucose from 5.6 mmo/L to 25 mmo/L every 8 h) or AGEs (150 mg/L, containing 5.6 mmo/L glucose).After 72 h treatment, Western blotting was used to determine the levels of the apoptotic protein cleaved-caspase-3.Other cells were trypsinized , washed with cold PBS and incubated with PI and Annexin V-FITC, then analyzed by flow cytometry to detect cell apoptosis .RE-SULTS:Diabetic patients had higher levels of fasting blood glucose (FBG), 2-hour postprandial blood glucose (2 h PBG) and glycosylated hemoglobin A1c (HbA1c), and longer wound duration.The protein level of cleaved caspase-3 was signifi-cantly higher in diabetic group , suggesting that apoptosis was increased in diabetic skin tissues .TUNEL analysis showed that apoptotic index was higher in diabetic group compared with that in non-diabetic group (8.4%±1.5% vs 3.8%± 0.8%) , which further confirmed that cell apoptosis was increased in diabetic foot tissues .In human fibroblasts , the levels of cleaved caspase-3 in normal group , sustained high glucose group , fluctuant high glucose group and AGEs group were 0.80 ±0.13, 1.22 ±0.18, 1.46 ±0.32 and 1.83 ±0.25, respectively.The apoptotic rates detected by flow cytometry were 2.43%±0.19%, 2.89%±0.51%, 3.99% ±0.24% and 6.83% ±0.36%, respectively.Both the level of cleaved caspase-3 and the apoptotic rate in AGEs group were higher than those in normal glucose group and sustained high glucose group .CONCLUSION:Increased apoptosis in diabetic foot ulcers is one of the most important reasons for im-paired wound healing .As compared to sustained high glucose and glucose fluctuations , AGEs induce greater apoptosis in human fibroblast cells .

Objective To investigate the change of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the skin of diabetic rats, and to explore the potential role of MMP-9/TIMP-1. Methods Diabetic rats were induced with streptozotocin (STZ). Then all rats were maintained for 6 weeks. Routine pathological examination and immnnohistochemistry were made to reveal the histological and cytological appearances. RT-PCR and Western blotting were used to detect the expression of mRNA and protein of MMP-9 and TIMP-1 in the skin. Results Six weeks after STZ treatment, examination of HE-stained skin sections from normal and diabetic animals revealed that the epidermis and dermis layers were thinner in diabetic rats than those in control rats. The skin of diabetic rats showed features of atrophy such as disorganization of connective tissue fiber bundles and enlarged space between collagen fiber bundles. In contrast, thick bundles of connective tissue were observed in the dermis of normal rat skin. In normal skin, cells had a bipolar, spindle-shaped appearance in the thick collagen bundles, while in the skin of diabetic animals the interstitial cells had a rounded, shrunken and crenated appearance. The relative values of expression of MMP-9 in diabetic group were higher than those in normal group with significant difference, however, the relative values of expression of TIMP-I in diabetic group were lower than those in control group. Conclusion The changes in cutaneous histology and cytology appear earlier than skin wound. These "underlying disorders" may be associated with the imbalance of MMP-9/TIMP-1.

AIM: To evaluate the pulmonary function in patients with type 2 diabetes mellitus in order to identify whether the lung is a target organ of chronic pathologic changes in diabetes mellitus. METHODS: Pulmonary ventilation function and diffusion capacity were studied in 107 patients with type 2 diabetes mellitus and 61 healthy subjects matched for age and sex. Glycosylated hemoglobin (HbA1c), urine albumin excretion rate (AER), fundus examination and nerve conduction velocity were included as parameters of glycemic control and diabetic microangiopathies. RESULTS: Pulmonary ventilation function was similar in type 2 diabetic group and the control. Compared with the control, carbon monoxide diffusion capacity (DLCO) and DLCO corrected by alveolar volume (DLCO/VA) were significantly lower in type 2 diabetic group (P<0.05). DLCO and DLCO/VA were inversely correlated with microangiopathy score (r: -0.291, -0.324, respectively, P<0.01). Furthermore, DLCO/VA was negatively correlated with age and duration of diabetes mellitus (r: -0.269, -0.236, respectively, P<0.05). CONCLUSIONS: Pulmonary ventilation function is normal in patients with type 2 diabetes mellitus, but their diffusion capacity is impaired. It suggests that the lung may also be the target organ of the chronic pathologic changes of diabetes mellitus.

AIM:To investigate the imbalance between the expression of metalloproteinases (MMPs) and that of tissue inhibitors of metalloproteinase (TIMPs) during wound healing in diabetic rats. METHODS: Diabetic rats were induced with streptozotocin. All rats were maintained for 6 weeks. A full-thickness excisional wound was created on the back of each rat. Every group was randomly divided into 3 subgroups of 7 rats: 3 d group, 7 d group, 14 d group and animals were killed at 3rd, 7th and 14th day. Routine pathological examination, Masson′s trichrome staining and immunohistochemistry were made to calculate the score of epidermal and dermal regeneration, granulation tissue thickness, angiogenesis, matrix density, and infiltrated cells at different time points. RT-PCR and Western blotting were used to detect the expression of mRNA and protein of MMP-9 and TIMP-1 in the skin at those time points. RESULTS: Six weeks after streptozotocin treatment, Three days after injury, the wound healing rate of normal rats was faster than that of diabetic rats. From 3rd to 14th day, there were a lot of fibroblast and macrophage in normal skin, while few such cells were observed in diabetic skin. The other histological scores in normal skin were higher than those in diabetic rats at 7th and 14th day. Both MMP-9 and TIMP-1 had minimally detectable levels before wounding but exhibited rapid, significantly large increases within 3 d after wounding. Subsequently, they showed a rapid decline by 14 d. The relative values of expression of MMP-9 mRNA and protein in diabetic group were higher than those in normal group at different time points. However, the values of TIMP-1 mRNA and protein in diabetic group were significantly lower than those in control group. Significant difference was observed between two groups with the ratio of MMP-9/TIMP-1, higher in diabetic group than that in normal group. CONCLUSION: Abnormal reepithelialization, angiogenesis, inflammatory cell infiltration, collagen fibers generation, granulation tissue deposition, seem to be the basic histopathology that delays wound healing. The imbalance between MMPs and TIMPs in diabetic skin tissue before and after injury may be one of the important reasons of these alterations of histopathology.

[Objective]To explore the relationship between peripheral arterial disease(PAD)and diabetic retinopathy(DR)in type 2 diabetes patients.[Methods]A total of 99 patients diagnosed with PAD were classified into grade 1-3 by their total scores of peripheral arterial stenosis assessed by color doppler ultrasound examinations,where the degree of stenosis 30% ~ 49% scored 0, 50%~99%scored 1,lumen occlusion(i.e. degree of stenosis 100%)scored 2,and therefore the total score 0-2 was categorized into Grade 1 ,3~4 into Grade 2 ,5~12 into Grade 3. The bilateral anterior tibial artery ,posterior tibial artery and dorsalis pedis artery of these patients were analyzed. The presence of diabetic retinopathy(DR)was graded from retinal photographs using a standard protocol.[Results]Among 99 cases of type 2 diabetic patients with peripheral arterial disease ,58.6%of them were male with average age of 67.3 ± 7.9 years old. Patients of Grade 1,Grade2,Grade 3 lesion accounted for 45.4%,30.3%,24.2%,respectively. Age, gender,smoking history,SBP,DBP,BMI,FBG,TC,TG,LDL-C,HDL-C,HbA1C among 3 groups were not statistically signifi-cant. The associations of DM duration and HbA1C value were significantly larger in DR than in PAD. The proportion of DR patients increased with the severity degree of PAD(p for trend=0.004). Degree of stenosis Grade 2 and Grade 3 could be predictive for DR.[Conclusions]DR is associated with the severity degree of PAD in type 2 diabetes patients as evaluated by duplex ultrasonography.Degree of stenosis Grade 2 and 3 could be used for screening or finding DR. Strategies for optimum treatment and early prevention are needed.

Primary hypomagnesemia with secondary hypocalcemia(HSH) is a rare cause of hypoparathyroidism. This article presents a case of a 26-year-old male with recurrent generalized weakness and tetany, and a literature review of diagnosis and treatment of primary HSH. The biochemical tests revealed the patient had severe hypomagnesemia, mild hypocalcemia, hypokalemia, and hypoparathyroidism. Transient receptor potential melastatin-6(TRPM6) gene mutation were detected by gene test, which confirmed the diagnosis of primary HSH. The patient had been treated with long term oral magnesium supplementation, who remained asymptomatic during the follow-up. Primary HSH is a rare autosomal-recessive disorder caused by mutations in the TRPM6 gene which encoding a magnesium permeable channel expressed in the intestine and the kidney. The primary defect is impaired intestinal absorption of magnesium with secondary renal excretion, leading to a series of clinical symptoms. The treatment is mainly through lifelong magnesium supplementation.