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Lin Yang,1,2 Xiaoyu Pan,2 Zhenyu Pan,3 Haina Gao,4 Jiangli Ban,4 Shuchun Chen2,5,6 1Department of Internal Medicine, Hebei North University, Zhangjiakou, Hebei, People’s Republic of China; 2Department of Endocrinology, Hebei General Hospital, Shijiazhuang, Hebei, People’s Republic of China; 3Department of Internal Medicine, Shandong Medical College, Linyi, Shandong, People’s Republic of China; 4Department of Endocrinology, The Second Hospital of Shijiazhuang, Shijiazhuang, Hebei, People’s Republic of China; 5Department of Internal Medicine, Hebei Medical University, Shijiazhuang, Hebei, People’s Republic of China; 6Hebei Key Laboratory of Metabolic Diseases, Hebei General Hospital, Shijiazhuang, Hebei, People’s Republic of ChinaCorrespondence: Shuchun Chen, Department of Internal Medicine, Hebei Medical University, People’s Republic of China, Tel +86 31185988406, Email chenshuc2014@163.comIntroduction: Obesity-induced inflammation and oxidative stress can cause damage to cardiomyocytes. Semaglutide has the potential to reduce glucose levels and weight, while hydroxysteroid dehydrogenase-like protein 2 (HSDL2) also plays a role in regulating lipid metabolism. This study aimed to investigate the expression of oxidative stress markers and HSDL2 in myocardium and serum under high-fat conditions, in order to elucidate the mechanism of obesity-induced myocardial injury and evaluate the impact of semaglutide on myocardial injury through HSDL2.Methods: Mouse models of obesity were established with semaglutide treatment. Palmitic acid-cultured mouse cardiomyocytes with HSDL2 knockout were used, as well as palmitic acid-induced high-fat environment models followed by semaglutide treatment. The levels of inflammatory and oxidative stress markers in serum and cardiomyocytes were measured. Additionally, the expression of HSDL2 and autophagy levels in different cell groups were assessed to evaluate the effect of semaglutide on high-fat diet-induced cardiomyocyte injury mediated by HSDL2.Results: Obesity increased oxidative stress, which was alleviated by intervention with semaglutide. Furthermore, semaglutide down-regulated HSDL2 expression in obese individuals. Moreover, palmitic acid-induced oxidative stress and autophagy were reduced when using cells with knocked out HSDL2 gene.Conclusion: These findings suggest that semaglutide may mitigate cardiomyocyte injury caused by a high-fat diet through regulation of HDLSDSLEP-1 expression. These discoveries are expected to unveil novel molecular mechanisms and provide new targets for clinical treatment.Keywords: obesity, inflammation, oxidative stress, semaglutide, HSDL2

Yi Zhu,1,2,* Meiling Chen,3,* Chuangzan Yang,1,2 Geng Lu,1,2 Sa Huang,1,2 Meili Chen,3 Yufei Wang,4 Junfeng Ban1– 3 1Guangdong Pharmaceutical University, Guangzhou, People’s Republic of China; 2The Innovation Team for Integrating Pharmacy with Entrepreneurship, Guangdong Pharmaceutical University, Guangzhou, People’s Republic of China; 3Guangdong Laboratory Animals Monitoring Institute, Guangdong Provincial Key Laboratory of Laboratory Animals, Guangzhou, People’s Republic of China; 4Analytical and Testing Center of Guangzhou University, Guangzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yufei Wang; Junfeng Ban, Email yufei_wang@gzhu.edu.cn; banjunfeng@163.comPurpose: Oral drug administration is the most common and convenient route, offering good patient compliance but drug solubility limits oral applications. Celecoxib, an insoluble drug, requires continuous high-dose oral administration, which may increase cardiovascular risk. The nanostructured lipid carriers prepared from drugs and lipid excipients can effectively improve drug bioavailability, reduce drug dosage, and lower the risk of adverse reactions.Methods: In this study, we prepared hyaluronic acid-modified celecoxib nanostructured lipid carriers (HA-NLCs) to improve the bioavailability of celecoxib and reduce or prevent adverse drug reactions. Meanwhile, we successfully constructed a set of FDA-compliant biological sample test methods to investigate the pharmacokinetics of HA-NLCs in rats.Results: The pharmacokinetic analysis confirmed that HA-NLCs significantly enhanced drug absorption, resulting in an AUC0-t 1.54 times higher than the reference formulation (Celebrex®). Moreover, compared with unmodified nanostructured lipid carriers (CXB-NLCs), HA-NLCs enhance the retention time and improve the drug’s half-life in vivo.Conclusion: HA-NLCs significantly increased the bioavailability of celecoxib. The addition of hyaluronic acid prolonged the drug’s in vivo duration of action and reduced the risk of cardiovascular adverse effects associated with the frequent administration of oral celecoxib. Keywords: nanostructured lipid carriers, celecoxib, HPLC-MS/MS, pharmacokinetics

Yiling Lin,1,2 Yingneng Zhang,1,2 Xia Cai,3 Huashen He,2 Chuangzan Yang,2 Junfeng Ban,1,2,4 Bohong Guo1 1School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, People’s Republic of China; 2The Innovation Team for Integrating Pharmacy with Entrepreneurship, Guangdong Pharmaceutical University, Guangzhou, People’s Republic of China; 3Guangdong Institute for Drug Control, Guangzhou, People’s Republic of China; 4Guangdong Provincial Key Laboratory of Advanced Drug Delivery, Guangdong Provincial Engineering Center of Topical Precise Drug Delivery System, Guangdong Pharmaceutical University, Guangzhou, People’s Republic of ChinaCorrespondence: Junfeng Ban; Bohong Guo, School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, Guangdong, 51006, People’s Republic of China, Tel +86 20 39352309, Email banjunfeng@163.com; bohong-g@163.comBackground: Delayed wound healing in skin injuries has become a significant problem in clinics, seriously affecting and even threatening life and health. Recently, research interest has increased in developing wound dressings containing bioactive compounds capable of improving outcomes for complex healing needs.Methods: In this study, Puerarin-loaded nanoparticles (Pue-NPs) were prepared using the cell-penetrating peptide-poly (lactic-co-glycolic acid) (CPP-PLGA) as a drug carrier by the emulsified solvent evaporation method. Then, they were added into poly (acrylic acid) to obtain a self-assembled nanocomposite hydrogels (SANHs) drug delivery system using the co-polymerization method. The particle size, zeta potential, and micromorphology of Pue-NPs were measured; the appearance, mechanical properties, adhesive strength, and biological activity of SANHs were performed. Finally, the potential of SANHs for wound healing was further evaluated in streptozotocin-induced diabetic mice.Results: Pue-NPs were regularly spherical, with an average particle size of 134.57 ± 1.42 nm and a zeta potential of 2.14 ± 0.78 mV. SANHs was colorless and transparent with a honeycomb-like porous structure and had an excellent swelling ratio (917%), water vapor transmission rate (3077 g·m− 2·day− 1), mechanical properties (Young’s modulus of 18 kPa, elongation at break of 307%), and adhesive strength (15.5 kPa). SANHs exhibited sustained release of Pue over 48h, with a cumulative release of 55.60 ± 6.01%. In vitro tests revealed that the SANHs presented a 92.22% antibacterial rate against Escherichia coli after 4h, and a 61.91% scavenging rate of 1.1-diphenyl-2-trinitrophenylhydrazine (DPPH) radical. In vivo experiments showed that SANHs accelerated wound repair by reducing the inflammatory response at the wound site, promoting angiogenesis, and facilitating epidermal regeneration and collagen deposition.Conclusion: In conclusion, we successfully prepared SANHs. Our results show that SANHs have excellent performance and improves wound healing in diabetic mice model, indicating that it can be used to develop an effective strategy for the treatment of diabetic wounds. Keywords: Puerarin-loaded nanoparticles, nanocomposite hydrogel, drug delivery, wound healing

Background: Bowel cancer is the most prevalent digestive system cancer and is the 4th largest cause of cancer-related death worldwide. In Iraq, colon and rectal cancer (CRC) is the 6th most common malignancy in males and the 5th in females. This cancer is sluggish in growth, which gives a window of opportunity to screen for both precursor lesions and early cancer. The Cluster of Differentiation 47 (CD47) protein is a type of transmembrane glycoproteins found on nearly all human cells, including non-hematopoietic and hematopoietic cells. CD47 promotes CRC growth by triggering angiogenesis and apoptosis of tumor cell. Objectives: To evaluate the immunohistochemical expression of (CD47) in various colorectal samples from Iraqi patients with CRC by immunohistochemistry (IHC) assay. Specimens and methods: A total of 45 paraffin-embedded CRC tissue specimens and clinical data were obtained from the Medical City Teaching Hospital in Baghdad and a number of private laboratories in Baghdad, Iraq. In addition, 30 control colon and rectum tissues with no significant pathology were collected from the Forensic Medicine Department for comparison purposes after taking the official approvals. Results: The results revealed a high expression of CD47 in CRC cases, but with no significant correlation with clinicopathological features. Also, the result of figures in this study revealed negative membranous expression of CD47 (score 0), strong membranous expression of CD47 (score 3), moderately membranous expression of CD47 (score 2), and weak membranous expression of CD47 (score 1) Conclusion: Patients with CRC had high CD47 expression, allowing tumor cells to modulate CD47SIRP inhibitory signaling and prevent immune cell attack.

Jianv Wang,1,* Hongye Liao,1,* Jieming Ban,2 Sen Li,2 Xia Xiong,1 Qingqing He,1 Xinyu Shi,2 Hongping Shen,2 Sijin Yang,2 Changzhen Sun,2 Li Liu1 1Department of Dermatology, the Affiliated Hospital of Southwest Medical University, Luzhou, 646000, People’s Republic of China; 2Drug Research Center of Integrated Traditional Chinese and Western Medicine, National Traditional Chinese Medicine Clinical Research Base, the Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou, 646000, People’s Republic of China*These authors contributed equally to this workCorrespondence: Changzhen Sun, Drug Research Center of Integrated Traditional Chinese and Western Medicine, National Traditional Chinese Medicine Clinical Research Base, the Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou, 646000, People’s Republic of China, Tel/Fax +86-136-9949-2406, Email sunchangzhen@swmu.edu.cn Li Liu, Department of Dermatology, the Affiliated Hospital of Southwest Medical University, Luzhou, 646000, People’s Republic of China, Tel/Fax +86-193-3860-9127, Email liuli@swmu.edu.cnBackground: Near-infrared cyanine dyes have high sensitivity and spatial resolution imaging capabilities, but they also have unavoidable drawbacks such as photobleaching, low water solubility, fluorescence quenching, and toxic side effects. As an effective biologic drug carrier, albumin combines with cyanine dyes to form albumin@dye nanoparticles. These nanoparticles can alleviate the aforementioned issues and are widely used in tumor imaging and photothermal therapy.Methods: Herein, a newly synthesized near-infrared dye IR-817 was combined with bovine serum albumin (BSA) to create BSA@IR-817 nanoparticles. Through the detection of fluorescence emission and absorption, the optimal concentration and ratio of BSA and IR-817 were determined. Subsequently, dynamic light scattering (DLS) measurements and scanning electron microscopy (SEM) were used for the physical characterization of the BSA@IR-817 nanoparticles. Finally, in vitro and in vivo experiments were conducted to assess the fluorescence imaging and photothermal therapeutic potential of BSA@IR-817 nanoparticles.Results: IR-817 was adsorbed onto the BSA carrier by covalent conjugation and supramolecular encapsulation, resulting in the formation of dispersed, homogeneous, and stable nanoparticles with a particle size range of 120– 220 nm. BSA@IR-817 not only improved the poor water solubility, fluorescence quenching, and toxic side effects of IR-817 but also enhanced the absorption and fluorescence emission peaks in the near-infrared region, as well as the fluorescence in the visible spectrum. In addition, BSA@IR-817 combined with laser 808 irradiation was able to convert light energy into heat energy with temperatures exceeding 50 °C. By creating a mouse model of subcutaneous melanoma, it was discovered that the tumor inhibition rate of BSA@IR-817 was greater than 99% after laser irradiation and that it achieved nearly complete tumor ablation without causing significant toxicity.Conclusion: Our research, therefore, proposes the use of safe and effective photothermal nanoparticles for the imaging, diagnosis, and treatment of melanoma, and offers a promising strategy for future biomedical applications.Keywords: albumin, cyanine dyes, near-infrared imaging, photothermal therapy, melanoma

Jiangli Ban,1,2 Xiaoyu Pan,2,3 Liqun Yang,2 Zhuoya Jia,2,3 Ruoxi Zhen,2,3 Xueqing Zhang,2,4 Shuchun Chen2,3 1Graduate School of Hebei North University, Zhangjiakou, People’s Republic of China; 2Department of Endocrinology, Hebei General Hospital, Shijiazhuang, People’s Republic of China; 3Graduate School of Hebei Medical University, Shijiazhuang, People’s Republic of China; 4Graduate School of North China University of Science and Technology, Tangshan, People’s Republic of ChinaCorrespondence: Shuchun Chen, Department of Endocrinology, Hebei General Hospital, 348 Heping West Road, Shijiazhuang, 050051, People’s Republic of China, Tel/Fax +86 31185988406, Email chenshuc2014@163.comPurpose: This study aimed to examine the correlation between fibrinogen/albumin (FAR) and diabetic peripheral neuropathy (DPN).Patients and Methods: A total of 342 patients were included and categorized into either the DPN group or the Non-DPN (NDPN) group based on their DPN status. The FAR index was determined by calculating the ratio of fibrinogen (FIB) to serum albumin (ALB), multiplied by 100. The participants were then divided into a High-FAR group and a Low-FAR group using the median FAR value as the threshold. Neurophysiological data were collected from the participants, which included motor conduction velocity (MCV) and sensory conduction velocity (SCV).Results: The DPN group displayed higher FAR levels [(DPN vs NDPN:6.72 (5.89,7.74) vs 5.94± 1.14], in addition to slower SCV and MCV data compared to the NDPN group. The high FAR group had a higher prevalence of DPN (78.9% vs 55.6%) (P< 0.05). There was a negative correlation between FAR and NCV, including bilateral median nerve SCV, left ulnar nerve SCV, bilateral median nerve MCV, bilateral common peroneal nerve MCV, bilateral tibial nerve MCV, and left ulnar nerve MCV. FAR was revealed to be an independent risk factor for the development of DPN in patients and demonstrated a greater predictive value for DPN development in Type 2 diabetes mellitus (T2DM) compared with FIB, HbA1c.Conclusion: The results suggest that monitoring FAR levels in patients with T2DM could identify those at higher risk for developing DPN, making the FAR index a valuable predictor of DPN development. Furthermore, since FAR has an inverse relationship with NCV, it stands to reason that high FAR levels may indicate nerve damage and slower conduction velocities. Thus, managing FAR could prove beneficial in both preventing and delaying the onset of DPN in T2DM patients.Keywords: type 2 diabetes mellitus, diabetic peripheral neuropathy, fibrinogen/albumin

Ruoxi Zhen,1,2 Jiangli Ban,2,3 Zhuoya Jia,1,2 Yanhui Liu,2,3 Zelin Li,1,2 Shuchun Chen1,2,4 1Graduate School, Hebei Medical University, Shijiazhuang, Hebei, People’s Republic of China; 2Department of Endocrinology, Hebei General Hospital, Shijiazhuang, Hebei, People’s Republic of China; 3Graduate School, Hebei North University, Zhangjiakou, People’s Republic of China; 4Hebei Key Laboratory of Metabolic Diseases, Shijiazhuang, Hebei, People’s Republic of ChinaCorrespondence: Shuchun Chen, Department of Endocrinology, Hebei General Hospital, 348 Heping West Road, Shijiazhuang, Hebei, 050051, People’s Republic of China, Tel +86 31185988406, Fax +86 31185988406, Email chenshuc2014@163.comPurpose: The objective of this research was to examine the relationship between non-HDL cholesterol/HDL cholesterol ratio (NHHR) and vitamin D in type 2 diabetes mellitus (T2DM).Patients and Methods: This study enrolled 617 T2DM participants. Participants were separated into two groups: no vitamin D deficiency and vitamin D deficiency. Participants were split into two categories: individuals who had a high NHHR and those with a low NHHR, with the median NHHR serving as the cut-off. Eventually, the study participants were classified into two groups by gender, which were further classified into vitamin D deficient and non-vitamin D deficient groups.Results: NHHR values were substantially greater in vitamin D deficient group than in the non-deficient group in both male and female T2DM patients (P< 0.05). The high NHHR group displayed substantially lower vitamin D levels than the low NHHR group [16.21 (12.55,21.35) vs 19.05 (14.59,24.07), P< 0.001]. NHHR was discovered to be negatively and independently associated with vitamin D levels, and there was no sex difference.Conclusion: For the first time, our research revealed a negative relationship between NHHR and vitamin D in patients with T2DM.Keywords: vitamin D, diabetes, type 2 diabetes, lipid ratio, non-HDL-C /HDL-C

Baohua Li,1,2,&ast; Geng Lu,1,2 Wenbin Liu,3,&ast; Liqi Liao,2 Junfeng Ban,1,2 Zhufen Lu1 1Guangdong Provincial Key Laboratory of Advanced Drug Delivery, Guangdong Provincial Engineering Center of Topical Precise Drug Delivery System, Guangdong Pharmaceutical University, Guangzhou, People’s Republic of China; 2The Innovation Team for Integrating Pharmacy with Entrepreneurship, Guangdong Pharmaceutical University, Guangzhou, People’s Republic of China; 3Guangdong Provincial Key Laboratory of Pharmaceutical Bioactive Substances, Guangdong Pharmaceutical University, Guangzhou, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Junfeng Ban; Zhufen Lu, School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, Guangdong, 51006, People’s Republic of China, Tel +86 20 39352362, Email banjunfeng@163.com; luzhufen@163.comIntroduction: The tight structure of the blood–brain barrier severely limits the level of drug therapy for central nervous system disorders. In this study, a novel composite delivery system combining nanocarrier and microneedle technology was prepared to explore the possibility of transdermal delivery of drugs to work in the brain.Methods: Nanoparticle solutions containing paroxetine and rhodamine-B were prepared using PLGA as a carrier by the emulsification-solvent volatilization method. Then, they were mixed with hyaluronic acid and the PLGA nanoparticulate-based microneedle system (Rh-NPs-DMNs) was prepared by a multi-step decompression-free diffusion method. The particle size, zeta potential, and micromorphology of the nano solution were measured; the appearance, mechanical strength, dissolution properties, and puncture effect of the Rh-NPs-DMNs were evaluated; also, it was evaluated for in vivo live imaging properties and in vitro skin layer transport and distribution properties.Results: The mean particle size of Rh-NPs was 96.25 ± 2.26 nm; zeta potential of 15.89 ± 1.97 mV; PDI of 0.120 ± 0.079. Rh-NPs-DMNs had a high needle content of 96.11 ± 1.27% and a tip height of 651.23 ± 1.28 μm, with excellent mechanical properties (fracture force of 299.78 ± 1.74 N). H&E skin tissue staining showed that Rh-NPs-DMNs produced micron-sized mechanical pores approximately 550 μm deep immediately after drug administration, allowing for efficient circulation of the drug; and the results of in vivo imaging showed that Rh-B NPs DMNs had a faster transport rate than Rh-B DMNs, with strong fluorescent signals in both brain (P< 0.01) and hippocampus (P< 0.05) 48 h after drug administration.Conclusion: Nanoparticles can prolong blood circulation time and intracerebral retention time and have certain brain-targeting properties due to their excellent physical properties. The use of microneedle technology combined with nanocarriers provides new ideas for delivery systems for the treatment of central neurological diseases.Graphical Abstract: Keywords: soluble microneedle, PLGA nanoparticles, skin transfer, central nervous system

Xiaoyu Pan,1,2 Zhuoya Jia,1,2 Ruoxi Zhen,1,2 Lin Yue,3 Shu Niu,1,4 Jiangli Ban,2 Shuchun Chen1,2 1Department of Internal Medicine, Hebei Medical University, Shijiazhuang, Hebei, People’s Republic of China; 2Department of Endocrinology, Hebei General Hospital, Shijiazhuang, Hebei, People’s Republic of China; 3Department of Endocrinology, The Third Hospital of Shijiazhuang, Shijiazhuang, Hebei, People’s Republic of China; 4Department of Endocrinology, Shijiazhuang People’s Hospital, Shijiazhuang, Hebei, People’s Republic of ChinaCorrespondence: Shuchun Chen, Department of Endocrinology, Hebei General Hospital, Shijiazhuang, People’s Republic of China, Tel/Fax +86 31185988406, Email chenshuc2014@163.comPurpose: Studies have shown that atherosclerotic plaques are associated with changes in the microbial composition of the intestinal flora and obesity, and that the small intestine plays an irreplaceable role in regulating intestinal flora homeostasis, but the role of the small intestine in the development of obesity-related atherosclerosis remains understudied. Therefore, this study explores the role of the small intestine in obesity-induced atherosclerosis and its molecular mechanisms.Methods: In the GSE59054 data, small intestine tissue samples from 3 normal and 3 obese mice were analyzed using bioinformatics methods. Screening for differentially expressed genes (DEGs) using the GEO2R tool. The DEGs were next processed for bioinformatics analysis. We constructed an obese mouse model and measured aortic arch pulse wave velocity (PWV). Aortic and small intestine tissues were stained with hematoxylin-eosin (HE) to observe pathological changes. Finally, immunohistochemistry was performed to verify the expression of small intestinal proteins.Results: We identified a total of 122 DEGs. Pathway analysis revealed that BMP4, CDH5, IL1A, NQO1, GSTM1, GSTA3, CAV1 and MGST2 were mainly enriched in the Fluid shear stress and atherosclerosis pathway. In addition, BMP4, NQO1 and GSTM1 are closely related to atherosclerosis. Ultrasound and pathological findings suggest the presence of obesity atherosclerosis. Immunohistochemistry verified high expression of BMP4 and low expression of NQO1 and GSTM1 in obese small intestine tissues.Conclusion: The altered expression of BMP4, NQO1 and GSTM1 in small intestine tissues during obesity may be related to atherosclerosis, and Fluid shear stress and atherosclerosis pathway may be the molecular mechanism of their role.Keywords: obesity, differentially expressed genes, atherosclerosis, small intestine, bioinformatics analysis

Xiaoyu Pan,1,2 Xueqing Zhang,2 Jiangli Ban,2 Lin Yue,3 Lin Ren,2 Shuchun Chen1,2 1Department of Internal Medicine, Hebei Medical University, Shijiazhuang, Hebei, People’s Republic of China; 2Department of Endocrinology, Hebei General Hospital, Shijiazhuang, Hebei, People’s Republic of China; 3Department of Endocrinology, the Third Hospital of Shijiazhuang, Shijiazhuang, Hebei, People’s Republic of ChinaCorrespondence: Shuchun Chen, Department of Internal Medicine, Hebei Medical University, People’s Republic of China, Tel +86 31185988406, Email chenshuc2014@163.comBackground: Neutrophils and high-density lipoprotein cholesterol (HDL-C) are significantly linked to cardiovascular disease (CVD). This study investigates the correlation of neutrophil count to HDL-C ratio (NHR) with cardiac ultrasound parameters and cardiovascular risk in healthy populations.Materials and Methods: Firstly, NHR was calculated based on neutrophils and HDL-C. Then, the differences in basic clinical characteristics and cardiac ultrasound parameters were compared between the high and low NHR groups, males and females. Subsequently, cardiovascular risk was predicted according to the Chinese 10-year ischemic cardiovascular disease (ICVD) risk assessment tool for people aged 35– 60 years. Finally, the correlation between NHR and cardiac ultrasound parameters and cardiovascular risk was calculated.Results: A total of 3020 healthy participants, 1879 males and 1141 females, were included. Participants in the high NHR group had significantly increased Aorta (AO), Left Atrium (LA), Right Atrium (RA), Right Ventricle (RV), End Systolic Diameter of Left Ventricle (ESD), End Diastolic Diameter of Left Ventricle (EDD), Main Pulmonary Artery (MPA), Right Ventricular Outflow Tract (RVOT), Interventricular Septum (IVS), Left Ventricular Posterior Wall (LVPW), and cardiovascular risk and decreased E/A values compared to those in the low NHR group. The same results were found in males participants compared to females. A total of 1670 participants underwent ICVD risk assessment tool. Cardiovascular risk was significantly higher in those with high NHR and in males than in those with low NHR and in females. Correlation analysis showed that NHR was positively correlated with AO, LA, RA, RV, ESD, EDD, MPA, RVOT, IVS, LVPW and cardiovascular risk, and negatively correlated with E/A values.Conclusion: Our study demonstrates that NHR is significantly associated with cardiac ultrasound parameters and cardiovascular risk in healthy populations. NHR may serve as a useful indicator for the early diagnosis and treatment of cardiovascular disease among healthy populations.Keywords: neutrophils, high-density lipoprotein cholesterol ratio, cardiovascular risk, cardiac ultrasound, cardiovascular disease

Xiaoyu Pan,1,2 Xueqing Zhang,2 Jiangli Ban,2 Lin Yue,3 Lin Ren,2 Shuchun Chen1,2 1Department of Internal Medicine, Hebei Medical University, Shijiazhuang, Hebei, People’s Republic of China; 2Department of Endocrinology, Hebei General Hospital, Shijiazhuang, Hebei, People’s Republic of China; 3Department of Endocrinology, The Third Hospital of Shijiazhuang, Shijiazhuang, Hebei, People’s Republic of ChinaCorrespondence: Shuchun Chen, Department of Internal Medicine, Hebei Medical University, Shijiazhuang, Hebei, People’s Republic of China, Tel +86 31185988406, Email chenshuc2014@163.comPurpose: To investigate the effect of high-fat diet on protein expression in mouse heart and aorta using proteomic techniques.Methods: A high-fat diet was used to construct an obese mouse model, and body weight was checked regularly. After the experiment, serum lipid and oxidative stress levels were measured. Proteomic detection of cardiac and aortic protein expression. Cardiac and aortic common differentially expressed proteins (Co-DEPs) were screened based on proteomic results. Subsequently, functional enrichment analysis and screening of key proteins were performed.Results: A high-fat diet significantly increased body weight in mice. Obese mice had considerably higher levels of TC, TG, LDL-C, ROS, and MDA. In the heart and aorta, 17 Co-DEPs were discovered. The results of functional analysis of these proteins indicated that they were mainly related to lipid metabolism. Ech1, Decr1, Hsd17b4, Hsdl2 and Acadvl were screened as key proteins. In mice, a high-fat diet causes lipid metabolism to become disrupted, resulting in higher levels of oxidative stress and lipid peroxidation products.Conclusion: Ech1, Decr1, Hsd17b4, Hsdl2 and Acadvl as cardiac and aortic Co-DEPs are closely related to lipid metabolism and may serve as potential diagnostic and therapeutic targets for obesity-induced cardiovascular disease.Keywords: high-fat diet, obesity, proteomics, cardiovascular disease, lipid

Jiangli Ban,1,2 Xuetong Zhao,3 Zhuoya Jia,2,4 Zelin Li,2,4 Shuchun Chen2,4,5 1Graduate School of Hebei North University, Zhangjiakou, People’s Republic of China; 2Department of Endocrinology, Hebei General Hospital, Shijiazhuang, People’s Republic of China; 3Baoding NO.1 Hospital, Baoding, People’s Republic of China; 4Graduate School of Hebei Medical University, Shijiazhuang, People’s Republic of China; 5Hebei Key Laboratory of Metabolic Diseases, Shijiazhuang, People’s Republic of ChinaCorrespondence: Shuchun Chen, Department of Endocrinology, Hebei General Hospital, 348 Heping West Road, Shijiazhuang, 050051, People’s Republic of China, Tel/Fax +86 31185988406, Email chenshuc2014@163.comPurpose: The relationship between atherogenic index of plasma (AIP) values and 25-hydroxyvitamin D (25[OH] D) was examined in type 2 diabetes mellitus (T2DM).Patients and Methods: Six hundred and ninety-eight T2DM patients were included. Patients were allocated to two groups, namely, the vitamin D-deficient and non-deficient groups (threshold of 20 ng/mL). The AIP was determined as log (TG [mmol/L] / HDL-C [mmol/L]). The patients were then allocated to two further groups according to the median AIP value.Results: AIP level in the vitamin D-deficient group was significantly higher than that in non-deficient group (P< 0.05). Patients with high AIP values had markedly reduced levels of vitamin D levels compared with those in the low-AIP group [15.89 (11.97, 20.29) VS 18.22 (13.89, 23.08), P< 0.001]. Patients in the high AIP group had a higher rate of vitamin D deficiency (73.3% VS 60.6%). It was found that AIP values were adversely and independently correlated with vitamin D levels. The AIP value was shown to independently predict vitamin D deficiency risk in T2DM patients.Conclusion: Patients with T2DM were shown to have an increased risk of vitamin D insufficiency when their AIP levels were low. This suggests that AIP is associated with vitamin D insufficiency in Chinese patients with type 2 diabetes.Keywords: vitamin D, diabetes mellitus, type 2 diabetes, atherogenic index of plasma

Xing Chen,1 Shuchun Chen,2,3 Qingjuan Ren,3 Shu Niu,3 Xiaoyu Pan,3 Lin Yue,3 Zelin Li,3 Ruiyi Zhu,3 Zhuoya Jia,3 Xiaoyi Chen,3 Ruoxi Zhen,3 Jiangli Ban3 1Department of Nephrology, Hebei General Hospital, Shijiazhuang, 050051, People’s Republic of China; 2Department of Endocrinology, Hebei General Hospital, Shijiazhuang, 050051, People’s Republic of China; 3Department of Internal Medicine, Hebei Medical University, Shijiazhuang, 050017, People’s Republic of ChinaCorrespondence: Shuchun Chen, Department of Endocrinology, Hebei General Hospital, 348 Heping West Road, Shijiazhuang, Hebei, 050051, People’s Republic of China, Tel/Fax +86 311 85988406, Email chenshuchunwork88@163.comPurpose: Semaglutide, a new long-acting glucagon-like peptide-1 analogue, has shown benefits for renal diseases, but its direct role on kidney metabolism under obesity remains unclear. The study aims to elucidate the protective effect and metabolic modulation mechanism of semaglutide on obesity-related kidney injury.Methods: Male C57BL/6J mice were divided into control and obesity groups. Mice in the obesity group had a high-fat diet and were treated with or without semaglutide (30nmol/kg/day). The study assayed blood biochemistry and then evaluated renal pathological injury through Periodic Acid-Schiff staining and electron microscopy. Metabolomics was utilized to analyze obesity-related metabolites in kidney samples.Results: Semaglutide significantly improved glucose homeostasis, insulin resistance, and kidney injury in obese mice. We successfully identified 377 altered metabolites (P< 0.05). It was suggested that semaglutide directly improved oxidative stress and inflammation-related metabolites such as nicotinamide adenine dinucleotide (NAD+) and adenosine in the kidney of obese mice, which have not been documented in obesity-related kidney injury. Relevant enriched pathways were included phospholipids and lysophospholipids metabolism, purine metabolism, NAD+ metabolism, and insulin resistance-related metabolism. They could serve as potential targets for intervention of obesity-related kidney injury.Conclusion: Our study revealed the metabolomics-based renoprotective mechanism of semaglutide in obese mice for the first time. The innovation lied in the identified metabolites such as NAD+ and adenosine targeted by semaglutide, which have not been documented in obesity-related kidney injury. Semaglutide may be a promising therapy for obesity-related kidney diseases.Keywords: semaglutide, glucagon-like peptide-1, metabolomics, obesity, kidney

Xiaoyu Pan,1,2 Lin Yue,1,2 Jiangli Ban,2 Lin Ren,2 Shuchun Chen1,2 1Department of Internal Medicine, Hebei Medical University, Shijiazhuang, People’s Republic of China; 2Department of Endocrinology, Hebei General Hospital, Shijiazhuang, People’s Republic of ChinaCorrespondence: Shuchun Chen, Department of Endocrinology, Hebei General Hospital, Shijiazhuang, People’s Republic of China, Tel +86 31185988406, Fax +86 31185988406, Email chenshuc2014@163.comPurpose: Using proteomics to study the effect of semaglutide on cardiac protein expression in obese mice. Assessment of the effect of semaglutide on cardiac function in obese mice.Materials and Methods: The mice were randomly divided into three groups: the control group (WC), the high-fat group (WF), and the high-fat diet with semaglutide intervention group (WS). Serum samples were collected, and lipids, blood glucose, inflammatory and oxidative stress markers, and cardiac ultrasound, were examined. The cardiac weight of each group of mice was measured, and pathological alterations were examined. Inflammation and oxidative stress levels in heart tissue were evaluated. The labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform was used to find differentially expressed proteins (DEPs) and screen for related pathways and key proteins in a proteomics study.Results: Semaglutide greatly alleviated obesity-induced lipid metabolism abnormalities, improved cardiac ventricular wall thickening, and significantly reduced myocardial collagen content in obese mice. Semaglutide significantly reduces obesity-induced inflammation and oxidative stress. There were 64 DEPs in the WF/WC group, with 39 upregulated proteins and 25 downregulated proteins. The WS/WC group, on the other hand, had 83 DEPs, including 57 upregulated and 26 downregulated proteins. Following functional analysis, DEPs were shown to be largely associated with lipid metabolism and peroxisomes. Apolipoprotein A-II, catalase, diazepam-binding inhibitor, paraoxonase-1, and hydroxysteroid 17-dehydrogenase-4 were all upregulated in the WF group but significantly downregulated in the WS group. A high-fat diet increases the expression of lipid synthesis and transport proteins while increasing inflammation and oxidative stress damage.Conclusion: Semaglutide decreases lipid synthesis alleviates inflammation and oxidative stress and prevents lipid peroxidation and cardiac impairment.Keywords: semaglutide, lipid metabolism, DEPs, obesity, inflammation, oxidative stress

Xiaoyu Pan,1,2 Lin Yue,3 Lin Ren,2 Jiangli Ban,2 Shuchun Chen1,2 1Department of Internal Medicine, Hebei Medical University, Shijiazhuang, Hebei, People’s Republic of China; 2Department of Endocrinology, Hebei General Hospital, Shijiazhuang, Hebei, People’s Republic of China; 3Department of Endocrinology, the Third Hospital of Shijiazhuang, Shijiazhuang, Hebei, People’s Republic of ChinaCorrespondence: Shuchun Chen, Department of Internal Medicine, Hebei Medical University, 361 Zhongshan East Road, Shijiazhuang, Hebei, 050017, People’s Republic of China, Tel +86 31185988406, Email chenshuc2014@163.comPurpose: To investigate the correlation between Triglyceride-glucose (TyG) index and liver function parameters in healthy obese civil servants in Shijiazhuang, China.Materials and Methods: This was an outpatient-based cross-sectional study in which 6452 participants were recruited. A total of 784 participants were analyzed according to inclusion and exclusion criteria. A TyG index was calculated based on fasting glucose and triglycerides. All patients were divided into a high TyG index group and a low TyG index group, using the median TyG index as a cut-off. Finally, patients were further divided into two subgroups: males and females.Results: While AST/ALT and direct bilirubin levels were lower in the high TyG index group compared to the low TyG index group, ALT, AST, total protein, and albumin levels were greater. Particularly in male participants, TyG index was inversely connected with AST/ALT and direct bilirubin levels and favorably correlated with ALT, AST, total protein, and albumin levels. These connections persisted in the multilinear regression study even after adjusting for confounding variables.Conclusion: This study describes the correlation between TyG index and liver function parameters in obese populations without co-morbid diseases, providing a new idea for early interventional treatment in this group.Keywords: TyG index, obesity, NAFLD, ALT, AST

The complex interplay between H. pylori and autophagy was elucidated in gastritis in term of host defense and survival of microbe. However, H. pylori frequently succeed in their survival and develops more aggressive tissue damage by different ways. A total of 80 gastritis patients undergoing gastroscopy during September, 2013 to August, 2014. Formalin fixed paraffin embedded gastric tissue were prepared for immunohistochemistry evaluation of ATG16L1, LC3C and vacA protein expression. Rapid urease test was used to discriminate between H. pylori or none H. pylori gastritis. Xenophagy index was elevated among both urease and IgG positive cases (29.86% and 27.13%) compared with negative cases (13.43% and 17.6% respectively (p

Background: Identifying predictors of the probabilities of conception related to the timing and frequency of intercourse in the menstrual cycle is essential for couples attempting pregnancy, users of natural family planning methods, and clinicians diagnosing for possible causes of infertility. The aim of this study is to estimate the days in which the likelihood of conception happened by using first trimester ultrasound fetal biometry in natural cycles and spontaneous pregnancy, and to explore some factors that may affect them. Materials and Methods: This study is retrospective cohort study, with random sampling. It involved 60 pregnant ladies at first trimester; the date of conception was estimated using: i. Crown-rump length biometry (routine ultrasound examinations were performed at a median of 70 days following Last menstrual period or equivalently 10 weeks), ii. Date of last menstrual cycle. Only women with previous infertility and now conceiving naturally with a certain date of Last menstrual period were selected. Results: The distribution of conception showed a sharp rise from day 8 onwards, reaching its maximum at day 13 and decreasing to zero by day 30 of Last menstrual period. The older and obese women had conceive earlier than younger women but there was insignificants difference between the two groups (P > 0.05). According to the type of infertility, the women with secondary infertility had conceived earlier than those with primary infertility. There was a significant difference between the two groups (P < 0.05). Conclusion: Day specific of conception may be affected by factors such as age, BMI, and type of infertility. This may be confirmed by larger sample size in metacentric study.

Objective: To investigate occur the histomatrical alternations in placental terminal villi and their vessels of Iraqi diabetes (gestational diabetes and overt diabetes) and normal pregnancies were born male and female neonates. Methods: The hitometrical study of male and female terminal villi of 68 placentae in maternal diabetes mellitus (34 gestational diabetes, 34 overt diabetes (17 DM type 1 and 17 DM type 2) and 34 normal placentae. These placentae obtained from Department of Obstetrics and Gynaecology in three hospitals in Baghdad city. Results: The study showed that there is a statistically significant with higher diameter in central and peripheral sections of the terminal villi and their vessels in male neonate. Moreover, terminal villous diameter in central and peripheral sections of placentae in both male and female neonates appeared highly significant differences (P≤0.001) between DM type 1 and DM type 2, but fetal blood vessels in this terminal villous did not record any significant difference (P>0.05). Conclusions: The current results concluded that increasing in diameters terminal villous and their fetal blood vessels of central and peripheral sections of placentae recorded increasing diameters of GDM peripheral sections of placentae in male neonate in comparison to placental sections of other study groups.

Junfeng Ban, Yan Zhang, Xin Huang, Guanghan Deng, Dongzhi Hou, Yanzhong Chen, Zhufen Lu Guangdong Provincial Key Laboratory of Advanced Drug Delivery Systems, Guangdong Pharmaceutical University, Guangzhou, People’s Republic of China Abstract: Drug delivery carriers can maintain effective therapeutic concentrations in the eye. To this end, we developed lipid nanoparticles (L/NPs) in which the surface was modified with positively charged chitosan, which engaged in hydrogen bonding with the phospholipid membrane. We evaluated in vitro corneal permeability and release characteristics, ocular irritation, and drug dynamics of modified and unmodified L/NPs in aqueous humor. The size of L/NPs was uniform and showed a narrow distribution. Corneal permeation was altered by the presence of chitosan and was dependent on particle size; the apparent permeability coefficient of dexamethasone increased by 2.7 and 1.8 times for chitosan-modified and unmodified L/NPs, respectively. In conclusion, a chitosan-modified system could be a promising method for increasing the ocular bioavailability of unmodified L/NPs by enhancing their retention time and permeation into the cornea. These findings provide a theoretical basis for the development of effective drug delivery systems in the treatment of ocular disease. Keywords: ocular drug delivery system, local bioavailability, dexamethasone, eye drop administration

Background and Objective:Determination of HER2 gene is crucial in breast carcinoma management and prognosis, as HER2 alterations are linked to a shorter disease-free period, overall survival and resistance to tamoxifen anti-estrogen therapy and other chemotherapy regimens, regardless of the nodal or hormone receptor status. This study aimed to estimate HER2 gene status of infiltrative mammary cancer cases with immunohistochemically equivocal (2+) score using Silver DNA in Situ Hybridization(SISH) technique and to investigate its association with clinicopathological variables. Methods: The study included 52 formalin-fixed paraffin embedded tissue blocks from female patients with invasive breast carcinoma with score of 2+ (equivocal) HER2 immunohistochemistry. All cases were studied by silver DNA in situ hybridization technique (SISH) for the determination of the amplified HER2 DNA. Results: TheSISH technique showed that HER2 gene was not amplified in 33 cases out of 52 (63.5%); while the rest of 19 cases (36.5%) revealed amplified gene status.According to age, HER2 gene status reported non-significant difference in the age groups between cases with amplified and non-amplified gene status (P=0.173). There was a significant negative association between positive Estrogen (ER) and Progesterone (PR) status and HER2 gene amplification (P= 0.002 and 0.017, respectively). Conclusion: More than half of breast carcinoma cases with equivocal HER2 immunoreactivity showed non-amplified gene status; this needs to be considered by oncologists in their management planning of breast cancer. Amplified HER2 gene is significantly associated with negative ER and PR status that affects patients’ management protocols and future outcome of the disease.

Background/Aim: Diagnosis of Hirschsprung's disease (HD) can be hard and requires good experience, principally for pathologists who infrequently encounter the disease. However, diagnosis is not always possible with hematoxylin and eosin (H and E) because staining has limitations in the identification of immature ganglion cells in neonates and the submucosal area. Aim: To assess the diagnostic role of calretinin immunostaining in HD in comparison to neuron-specific enolase. Patients and Methods: Formalin-fixed paraffin tissue blocks of full-thickness distal colonic and rectal biopsies for 48 patients who clinically presented with symptoms suspicious for HD were collected for the period from December 2012 to January 2016. All biopsies were already studied by routine H and E histopathological examination for the presence or absence of ganglion cells. Further confirmation of ganglion cells and nerve fibers was performed by immunohistochemical study for neuron-specific enolase and calretinin, respectively, in a private pathology laboratory. Results: According to the histopathological assessment, cases with absent ganglionic cells were considered to be HD, which comprised 40 cases out of the total 48 cases. The mean age for HD cases was 19.43 months. The male-to-female ratio in HD cases was 2.34:1. All HD cases showed negative expression of calretinin in small nerve fibers of the lamina propria, musularis mucosae, and submucosa, and negative expression of neuron-specific enolase in ganglionic cells. The sensitivity, specificity, positive predictive value, and negative predictive values for both the markers in the confirmation of diagnosis of HD were all 100%. Conclusion: Calretinin immunostaining, similar to that of neuron-specific enolase, is a highly sensitive and specific diagnostic aid to histopathological examination in suspected HD.

Background/Aim: Colorectal cancer (CRC) ranks sixth among the most common 10 cancers in Iraq. It is a foremost public health dilemma and there is improved interest in understanding the fundamental principles of its molecular biology. DNA methylation in cancer has become the issue of passionate investigation. As compared with normal cells, the malignant cells show major disruptions in their DNA methylation patterns. We aimed to assess the association of global DNA hypomethylation in colonic adenomas and carcinomas of Iraqi patients, measured by immunohistochemistry of 5-methylcytosin, with different clinicopathological variables. Patients and Methods: Thirty tissue paraffin blocks from patients with colorectal adenomas, 30 tissue paraffin blocks from patients with colorectal adenocarcinomas, and 30 samples of apparently normal colonic tissue taken from autopsy cases as a control group were included in the present study. From each block, two sections of 5 μm thickness were taken, one section was stained with Hematoxylin and Eosin for revision of histopathological diagnosis and one section was immunohistochemically stained for 5-methylcytosine (5mC) and digitally analyzed by AperioImageScope software. Results: The mean digital value of 5mC immunohistochemical expression was sequentially decreased during neoplastic progression from normal colonic tissue into adenoma and then to carcinoma. The mean digital value of 5mC expression was significantly lower in large size adenomas (≥1 cm), and those with severe dysplasia. Concerning carcinoma cases, 5mC expression was significantly lower in stage C2. Conclusions: The immunohistochemical evaluation of 5mC yields refined information on colorectal tumor biology in adenoma and carcinoma. Global DNA hypomethylation reflected by low immunohistochemical expression of 5-mC is associated with advanced colorectal adenomatous polyps suggesting that it is an early event in colorectal carcinogenesis. Also this hypomethylation can reflect bad prognosis of patients with colorectal cancer by its correlation to higher tumor stage.

Background: According to the Iraqi Cancer Registry, renal carcinoma is the 13th most common carcinoma, with 798 registered cases in 2020 and 418 deaths. Methods: This is a cross sectional retrospective study that includes the analysis of 116 randomly selected sample of pediatric patients (74 nephrectomies and 42 biopsies) sent to the Teaching Laboratories of AL-Emamain AL-Kadhmain city (AS), Baghdad medical city, and private laboratories from January 2019 to January 2023. Results: Histopathological diagnosis revealed a ratio from malignant to benign of 7.4:1. Wilm’s tumor was the most common malignant tumor (65.5%), while multicystic renal dysplasia was the most common benign tumor (7.8%). Abdominal pain was the most common clinical feature (67.2%), followed by abdominal mass (20.7%). Regarding malignant cases, Pathological stage I was the most common (39.0%). As for the histopathological subtype, triphasic type was the most common (57.1%), followed by the biphasic type (10.5%). Conclusions, Malignant tumors make up the vast majority of renal tumors. Wilm tumor is the predominant malignant tumor. Fortunately, most cases of Wilm tumors exhibited an early stage and favorable histology. Neuroblastoma was the second most common malignant tumor. The abdominal lump is the most common presentation of pediatric renal tumor. [ABSTRACT FROM AUTHOR]

Background/Aim: To evaluate the immunohistochemical expression of matrix metalloproteinase-7 (MMP-7) in colorectal adenomas, and to correlate this expression with different clinicopathological parameters. Patients and Methods: The study was retrospectively designed. Thirty three paraffin blocks from patients with colorectal adenoma and 20 samples of non-tumerous colonic tissue taken as control group were included in the study. MMP-7 expression was assessed by immunohistochemistry method. The scoring of immunohistochemical staining was conducted utilizing a specified automated cellular image analysis system (Digimizer). Results: The frequency of positive immunohistochemical expression of MMP-7 was significantly higher in adenoma than control group (45.45% versus 10%) (P value < 0.001). Strong MMP-7 staining was mainly seen in adenoma cases (30.30%) in comparison with control (0%) the difference is significant ( P < 0.001). The three digital parameters of MMP-7 immunohistochemical expression (Area (A), Number of objects (N), and intensity (I)) were significantly higher in adenoma than control. Mean (A and I) of MMP-7 showed a significant correlation with large sized adenoma (≥ 1cm) ( P < 0.05), also a significant positive correlation of the three digital parameters (A, N, and I) of MMP-7 expression with villous configuration and severe dysplasia in colorectal adenoma had been identified ( P < 0.05). Conclusion: MMP-7 plays an important role in the growth and malignant conversion of colorectal adenomas as it is more likely to be expressed in advanced colorectal adenomatous polyps with large size, severe dysplasia and villous histology. The use of automated cellular image analysis system (Digmizer) to quantify immunohistochemical staining yields more consistent assay results, converts semi-quantitative assay to a truly quantitative assay, and improves assay objectivity and reproducibility.

Background/Aim: To evaluate the immunohistochemical expression of proliferating cell nuclear antigen (PCNA) and CD34 in colorectal adenomas and carcinomas, and to correlate this expression with different clinicopathologic parameters. Materials and Methods: The study was retrospectively designed. A total of 86 tissue samples, including 33 paraffin blocks from patients with colorectal adenomas, 33 paraffin blocks from patients with colorectal adenocarcinomas, and a control group of 20 samples of nontumerous colonic tissue, were included in the study. From each block, 3 sections of 5 ΅m thickness were taken, 1 section was stained with hematoxylin and eosin (H and E) and the other 2 sections were stained immunohistochemically for PCNA and CD34. Scoring of the immunohistochemical staining was performed using a specified automated cellular image analysis system (Digimizer). Results: PCNA expression was significantly increased in a sequence of normal mucosa-adenoma-carcinoma. It was significantly higher in adenomas ≥ 1 cm and those with severe dysplasia, and it showed a significant positive correlation with grade and lymph node involvement in colorectal carcinoma. CD34 showed significantly higher expression in carcinoma than adenoma and in adenoma than in the control group. CD34 expression showed a significant correlation with adenomas carrying severe dysplasia and large-sized adenomas (≥1cm). It was significantly correlated with tumor grade, lymphovascular invasion, and lymph node involvement in colorectal carcinoma. Conclusion: PCNA plays an important role in colorectal neoplastic progression and can be utilized as ancillary marker for the risk of malignant transformation in colorectal adenomas as it correlates with high grade dysplasia and size. Intratumoral quantification of the mean (A and N) of CD34 in colorectal carcinoma reflects the grade of tumors and can predict lymph node involvement and lymphovascular invasion, to make a useful additional prognostic factor.