ObjectiveTo explore the effect of oral sodium butyrate on skeletal muscle atrophy in CT26 tumor mice through the gut microbiota-skeletal muscle axis and its potential mechanism. MethodsSixty SPF BALB/c male mice aged 8 weeks were randomly divided into a normal control group (NC, n=18) and a ABX-depleted group (ABX, n=42). The ABX mice were pretreated with a quadruple antibiotic cocktail via oral gavage (0.2 ml per administration, once daily, 6 d per week, for 2 weeks), whereas NC received an equal volume of sterile water. The quadruple antibiotic cocktail consisted of metronidazole (1 g/L), vancomycin (0.5 g/L), ampicillin (1 g/L), and gentamicin (1 g/L). Following successful pretreatment, six mice from each group were randomly selected for gut microbiota sequencing analysis and designated as the Abx group and the NC0 group, respectively. Theremaining mice in ABX were subcutaneously inoculated in the dorsum with 0.2 ml of CT26 cell suspension (at a cell density of 1×10⁷/ml). Then these mice were randomly allocated into three subgroups: a control tumor bearing model group (0_NaB, n=12), a tumor-bearing model group receiving low-dose oral sodium butyrate (L_NaB, n=12), a tumor-bearing model group receiving high-dose oral sodium butyrate (H_NaB, n=12). And mice in NC were inoculated at the same site with 0.2 ml of normal saline. The administration dose for L_NaB was 0.3 g/(kg·d), that for H_NaB was 0.5 g/(kg·d), while NC and 0_NaB were given the same volume of normal saline (0.2ml per time, once daily, 6 d per week, for 4 weeks). The general condition of mice was monitored, and forelimb grip strength gastrocnemius muscle mass and its muscle fiber cross-sectional area were measured for each group. The structural changes in gut microbiota were assessed by 16S rRNA sequencing of cecal contents. Pathological alterations in the intestinal wall were examined via HE staining. Serum and gastrocnemius muscle levels of TNF‑α, IL-6, IL-1β, and LPS were quantified using ELISA. The protein expression of ZO-1 and occludin in the small intestine, as well as proteins associated with the TLR4/MyD88/NF-κB signaling pathway in the gastrocnemius muscle, were detected by Western blot analysis. Results(1) The alpha-diversity in Abx was significantly lower than that in NC0 (P<0.01), a significant decrease of the mass and muscle fiber cross-sectional area of the gastrocnemius (P<0.01), with the majority of gut microbiota being effectively depleted. (2) Compared with NC, the subcutaneous tumors of mice in 0_NaB were prominent, a significant increase of the mass and muscle fiber cross-sectional area of the gastrocnemius, accompanied by a significant decrease in body weight at the end of the 3th and 4th week (P<0.05), and a significant weakening of the forelimb grasping strength at the 5th and 6th week (P<0.01). Compared with 0_NaB, the tumor mass of mice in L_NaB and H_NaB showed a significant decreasing trend, and the grip strength of the forelimbs significantly increased at the 5th and 6th week (P<0.05, P<0.01). (3) Compared with 0_NaB, the Shannon and Observed species indices in α diversity of L_NaB and H_NaB were significantly increased (P<0.05). At the genus level, compared with 0_NaB, L_NaB exhibited a significant decrease in the relative abundance of Parasutterella (P< 0.01), while H_NaB showed significant reductions in the relative abundances of both Escherichia-Shigella and Parasutterella (P < 0.01). (4) Compared with 0_NaB, the small intestinal tissue structure in L_NaB and H_NaB was more intact, the infiltration of inflammatory cells was significantly reduced, and the capillaries were slightly dilated. The expression levels of ZO-1 and occludin proteins in L_NaB were significantly increased (P<0.01). (5) The LPS concentration in the gastrocnemius muscle and the protein expression levels of TLR4, MyD88, p-IκBα, and p-NF‑κB p65 in L_NaB and H_NaB were significantly lower than those in 0_NaB (P<0.05). The serum TNF‑α concentration in H_NaB and TNF-α concentration in the gastrocnemius muscle of the L_NaB and H_NaB were significantly lower than those in 0_NaB (P<0.05, P<0.01, P<0.01). ConclusionOral administration of NaB can improve gut microbiota α diversity, adjusting its composition, improving intestinal mucosal barrier function, reducing the LPS-induced pro-inflammatory response, and delaying skeletal muscle atrophy. The underlying mechanism may involve down regulation of TLR4/MyD88/NF-κB signaling in skeletal muscle.

Panax species are mostly valuable medicinal plants. While some species' seeds are sensitive to dehydration, the dehydration tolerance of seeds from other Panax species remains unclear. The phospholipase D(PLD) gene plays an important role in plant responses to dehydration stress. However, the characteristics of the PLD gene family and their mechanisms of response to dehydration stress in seeds of Panax species with different dehydration tolerances are not well understood. This study used seeds from eight Panax species to measure the germination rates and PLD activity after dehydration and to analyze the correlation between dehydration tolerance and seed traits. Bioinformatics analysis was also conducted to characterize the PnPLD and PvPLD gene families and to evaluate their expression patterns under dehydration stress. The dehydration tolerance of Panax seeds was ranked from high to low as follows: P. ginseng, P. zingiberensis, P. quinquefolius, P. vietnamensis var. fuscidiscus, P. japonicus var. angustifolius, P. japonicus, P. notoginseng, and P. stipuleanatus. A significant negative correlation was found between dehydration tolerance and seed shape(three-dimensional variance), with flatter seeds exhibiting stronger dehydration tolerance(r=-0.792). Eighteen and nineteen PLD members were identified in P. notoginseng and P. vietnamensis var. fuscidiscus, respectively. These members were classified into five isoforms: α, β, γ, δ, and ζ. The gene structures, subcellular localization, physicochemical properties, and other characteristics of PnPLD and PvPLD were similar. Both promoters contained regulatory elements associated with plant growth and development, hormone responses, and both abiotic and biotic stress. During dehydration, the PLD enzyme activity in P. notoginseng seeds gradually increased as the water content decreased, whereas in P. vietnamensis var. fuscidiscus, PLD activity first decreased and then increased. The expression of PLDα and PLDδ in P. notoginseng seeds initially increased and then decreased, whereas in P. vietnamensis var. fuscidiscus, the expression of PLDα and PLDδ consistently decreased. In conclusion, the dehydration tolerance of Panax seeds showed a significant negative correlation with seed shape. The dehydration tolerance in P. vietnamensis var. fuscidiscus and dehydration sensitivity of P. notoginseng seeds may be related to differences in PLD enzyme activity and the expression of PLDα and PLDδ genes. This study provided the first systematic comparison of dehydration tolerance in Panax seeds and analyzed the causes of tolerance differences and the optimal water content for long-term storage at ultra-low temperatures, thus providing a theoretical basis for the short-term and ultra-low temperature long-term storage of medicinal plant seeds with varying dehydration tolerances.
Seeds/metabolism* ; Panax/physiology* ; Plant Proteins/metabolism* ; Gene Expression Regulation, Plant ; Phospholipase D/metabolism* ; Plants, Medicinal/enzymology* ; Germination ; Multigene Family ; Water/metabolism* ; Dehydration ; Phylogeny

Medical institutions, with their clinical practice foundation and abundant human use experience data, have become important carriers for the inheritance and innovation of traditional Chinese medicine(TCM) and the "cradles" of the preparation of new TCM. To effectively promote the transformation of new TCM originating from the TCM clinical practice in medical institutions and establish an effective evaluation index system for the transformation of new TCM conforming to the characteristics of TCM, consensus experts adopted the literature research, questionnaire survey, Delphi method, etc. By focusing on the policy and technical evaluation of new TCM originating from the TCM clinical practice in medical institutions, a comprehensive evaluation from the dimensions of drug safety, efficacy, feasibility, and characteristic advantages was conducted, thus forming a comprehensive evaluation system with four primary indicators and 37 secondary indicators. The expert consensus reached aims to encourage medical institutions at all levels to continuously improve the high-quality research and development and transformation of new TCM originating from the TCM clinical practice in medical institutions and targeted at clinical needs, so as to provide a decision-making basis for the preparation, selection, cultivation, and transformation of new TCM for medical institutions, improve the development efficiency of new TCM, and precisely respond to the public medication needs.
Medicine, Chinese Traditional/standards* ; Humans ; Consensus ; Drugs, Chinese Herbal/therapeutic use* ; Surveys and Questionnaires

This study aims to explore the scientific connotation of sinking Rehmanniae Radix has the best quality and compare the quality between floating and sinking fresh Rehmanniae Radix samples. Ultra-performance liquid chromatography tandem quadrupole electrostatic field Orbitrap high-resolution mass spectrometry(UHPLC-Q-Orbitrap HRMS) was employed to detect the chemical components in floating and sinking fresh Rehmanniae Radix samples. The fingerprint of fresh Rehmanniae Radix was established by high performance liquid chromatography(HPLC), and four index components were determined simultaneously. The cluster analysis, principal component analysis(PCA), and orthogonal partial least squares-discriminant analysis(OPLS-DA) were conducted to compare the quality of floating and sinking fresh Rehmanniae Radix samples. An evaporative light-scattering detector was used to compare the content of five sugars. The extract yield and drying rate were determined, and the quality connotation of sinking Rehmanniae Radix has the best quality was explained by multiple indicators. A total of 41 components were preliminarily identified from fresh Rehmanniae Radix by UHPLC-Q-Orbitrap HRMS, including 7 iridoid glycosides, 9 phenylethanol glycosides, 6 amino acids, 4 sugars, 3 phenolic acids, 5 nucleosides, 3 organic acids, 1 ionone, 1 furan, 1 coumarin, and 1 phenylpropanoid. The results showed that the main chemical components were consistent between floating and sinking fresh Rehmanniae Radix. Nine common peaks were identified in the fingerprints of 15 batches of floating and sinking fresh Rehmanniae Radix samples, and the similarity of fingerprints was greater than 0.9. The cluster analysis, PCA, and OPLS-DA classified floating and sinking fresh Rehmanniae Radix sasmples into two categories, indicating differences in the quality between them. The total content of catalpol, rehmannioside D, ajugol, and verbascoside in sinking fresh Rehmanniae Radix samples was higher than that in floating samples of the same batch and specification, and the main differential component was catalpol. The total content of fructose, glucose, sucrose, raffinose, and stachyose in sinking fresh Rehmanniae Radix samples was higher than that in floating samples of the same batch and specification, and the main differential component was stachyose. The extract yield and drying rate of the sinking samples were higher than those of floating samples. This study preliminarily showed that floating and sinking fresh Rehmanniae Radix samples had the same components but great differences in the content of medicinal substance basis. The total content of four glycosides and five sugars, extract yield, and drying rate of sinking fresh Rehmanniae Radix samples is higher than that of floating samples of the same batch and specification. These findings, to a certain extent, explains the scientificity of sinking Rehmanniae Radix has the best quality recorded in ancient books and provide a reference for the quality control and clinical application of fresh Rehmanniae Radix.
Chromatography, High Pressure Liquid/methods* ; Drugs, Chinese Herbal/chemistry* ; Rehmannia/chemistry* ; Chemometrics ; Mass Spectrometry/methods* ; Quality Control ; Principal Component Analysis ; Plant Extracts

The chemical components of Carthami Flos were investigated by using macroporous resin, silica gel column chromatography, reversed-phase octadecylsilane(ODS) column chromatography, Sephadex LH-20, and semi-preparative high-performance liquid chromatography(HPLC). The planar structures of the compounds were established based on their physicochemical properties and ultraviolet-visible(UV-Vis), infrared(IR), high-resolution electrospray ionization mass spectrometry(HR-ESI-MS), and nuclear magnetic resonance(NMR) spectroscopic technology. The absolute configurations were determined by comparing the calculated and experimental electronic circular dichroism(ECD). Six flavonoid C-glycosides were isolated from the 30% ethanol elution fraction of macroporous resin obtained from the 95% ethanol extract of Carthami Flos, and identified as saffloquinoside F(1), 5-hydroxysaffloneoside(2), iso-5-hydroxysaffloneoside(3), isosafflomin C(4), safflomin C(5), and vicenin 2(6). Among these, the compounds 1 to 3 were new chalcone C-glycosides. The compounds 1, 2, 4, and 5 could significantly increase the viability of H9c2 cardiomyocytes damaged by oxygen-glucose deprivation/reoxygenation(OGD/R) at a concentration of 50 μmol·L~(-1), showing their good cardioprotective activity.
Glycosides/pharmacology* ; Flowers/chemistry* ; Drugs, Chinese Herbal/pharmacology* ; Carthamus tinctorius/chemistry* ; Chalcones/pharmacology* ; Animals

This study investigated the mechanism by which Jianpi Bushen Yiqi Decoction promotes acetylcholine receptor(AChR) clustering in myasthenia gravis through the Agrin/low-density lipoprotein receptor-related protein 4(LRP4)/muscle-specific receptor tyrosine kinases(MuSK) signaling pathway. A total of 114 female C57BL/6J mice were divided into the normal group, modeling group, and solvent control group. The normal group and the solvent control group were immunized with phosphate-buffered saline(PBS), while the modeling group was established as an experimental autoimmune myasthenia gravis(EAMG) model using the murine-derived AChR-α subunit R97-116 peptide fragment. After successful modeling, the mice were randomly assigned to the model group, the low-, medium-, and high-dose Jianpi Bushen Yiqi Decoction groups, and the prednisone group. After four weeks of continuous treatment, muscle strength was assessed using Lennon scores and grip strength tests. Immunofluorescence staining was conducted on differentiated C2C12 myotubes incubated with a drug-containing serum to observe the number of AChR clusters. The integrity of AChR on myofilaments in mouse gastrocnemius muscles was further assessed by immunofluorescence staining. Hematoxylin-Eosin(HE)staining was applied to examine pathological changes in the gastrocnemius muscles of EAMG mice treated with Jianpi Bushen Yiqi Decoction. Western blot was utilized to detect the expression of key proteins in the Agrin/LRP4/MuSK signaling pathway in both C2C12 myotubes and mouse gastrocnemius muscles. The results demonstrated that compared to the model group, the prednisone group exhibited a significant decrease in the body weights of mice, whereas no significant differences in the body weights of mice were observed among the low-, medium-, and high-dose Jianpi Bushen Yiqi Decoction groups. All treatment groups showed significantly improved grip strength and Lennon scores. Additionally, the formula promoted AChR clustering on myotubes and enhanced AChR integrity in gastrocnemius myofilaments and reduced inflammatory infiltration between muscle tissue and fibrous hyperplasia. Furthermore, Jianpi Bushen Yiqi Decoction upregulated the protein expression of AChRα1, Agrin, and p-MuSK in C2C12 myotubes and increased the protein expression of AChRα1, Agrin, MuSK, p-MuSK, LRP4, and docking protein 7(Dok-7)in gastrocnemius tissue. In conclusion, Jianpi Bushen Yiqi Decoction may promote AChR clustering by targeting key proteins in the Agrin/LRP4/MuSK signaling pathway, thereby improving neuromuscular junction function and enhancing muscle strength.
Animals ; Agrin/genetics* ; Mice ; Drugs, Chinese Herbal/administration & dosage* ; Signal Transduction/drug effects* ; Receptors, Cholinergic/genetics* ; Female ; Mice, Inbred C57BL ; Receptor Protein-Tyrosine Kinases/genetics* ; Neuromuscular Junction/metabolism* ; Myasthenia Gravis, Autoimmune, Experimental/physiopathology* ; Humans ; LDL-Receptor Related Proteins

This study aims to investigate the effect of Chaijin Jieyu Anshen Formula on the neuroinflammation of rats with insomnia complicated with depression through the regulation of triggering receptor expressed on myeloid cells 2(TREM2)/complement protein C1q signaling pathway. Rats were randomly divided into a normal group, a model group, a positive drug group, as well as a high, medium, and low-dose groups of Chaijin Jieyu Anshen Formula, with 10 rats in each group. Except for the normal group, the other groups were injected with p-chlorophenylalanine and exposed to chronic unpredictable mild stress to establish the rat model of insomnia complicated with depression. The sucrose preference experiment, open field experiment, and water maze test were performed to evaluate the depression in rats. Enzyme-linked immunosorbent assay was employed to detect serum 5-hydroxytryptamine(5-HT), dopamine(DA), and norepinephrine(NE) levels. Hematoxylin and eosin staining and Nissl staining were used to observe the damage in nucleus accumbens neurons. Western blot and immunofluorescence were performed to detect TREM2, C1q, postsynaptic density 95(PSD-95), and synaptophysin 1(SYN1) expressions in rat nucleus accumbens, respectively. Golgi-Cox staining was utilized to observe the synaptic spine density of nucleus accumbens neurons. The results show that, compared with the model group, Chaijin Jieyu Anshen Formula can significantly increase the sucrose preference as well as the distance and number of voluntary activities, shorten the immobility time in forced swimming test and the successful incubation period of positioning navigation, and prolong the stay time of space exploration in the target quadrant test. The serum 5-HT, DA, and NE contents in the model group are significantly lower than those in the normal group, with the above contents significantly increased after the intervention of Chaijin Jieyu Anshen Formula. In addition, Chaijin Jieyu Anshen Formula can alleviate pathological damages such as swelling and loose arrangement of tissue cells in the nucleus accumbens, while increasing the Nissl body numbers. Chaijin Jieyu Anshen Formula can improve synaptic damage in the nucleus accumbens and increase the synaptic spine density. Compared to the normal group, the expression of C1q protein was significantly higher in the model group, while the expression of TREM2 protein was significantly lower. Compared to the model group, the intervention with Chaijin Jieyu Anshen Formula significantly downregulated the expression of C1q protein and significantly upregulated the expression of TREM2. Compared with the model group, the PSD-95 and SYN1 fluorescence intensity is significantly increased in the groups receiving different doses of Chaijin Jieyu Anshen Formula. In summary, Chaijin Jieyu Anshen Formula can reduce the C1q protein expression, relieve the TREM2 inhibition, and promote the synapse-related proteins PSD-95 and SNY1 expression. Chaijin Jieyu Anshen Formula improves synaptic injury of the nucleus accumbens neurons, thereby treating insomnia complicated with depression.
Animals ; Male ; Rats ; Nucleus Accumbens/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Depression/complications* ; Membrane Glycoproteins/genetics* ; Rats, Sprague-Dawley ; Sleep Initiation and Maintenance Disorders/complications* ; Neurons/metabolism* ; Receptors, Immunologic/genetics* ; Signal Transduction/drug effects* ; Synapses/metabolism*

OBJECTIVE: To establish digital model of Ideberg typeⅡregional glenoid fracture anatomical plate with 3D metal printing technology. METHODS: The scapular imaging data of a 34-year-old healthy male volunteer were retrospectively selected. Mimics 15.01, NX 12.0 and other software were used to design Ideberg typeⅡ regional scapular fracture guide plate system. STL data were input into a metal 3D printer to print 1∶1 scapular model and anatomical plate of scapular pelvis with guide sleeve. The fit of the plate was tested in vitro and the accuracy of the screw position was evaluated by imaging. The printing time of scapular model, design of the nail path and making time of the anatomic guided plate were recorded. RESULTS: 3D metal-printed Ideberg typeⅡ guide plate for scapular fracture fitted well to 3D printed scapular model, the locking screw was oriented accurately, and X-ray and CT showed good screw position. The printing time of scapula model, time of nail path design and special-shaped anatomical guide plate production were 52.0, 15.0 and 320 min, respectively. CONCLUSION Anatomical plates based on 3D metal printing technology could achieve good adhesion of Ideberg typeⅡ regional fractures and precise screw placement, providing a new and accurate surgical method for the treatment of Ideberg typeⅡ glenoid fractures.
Humans ; Printing, Three-Dimensional ; Male ; Adult ; Bone Plates ; Fractures, Bone/diagnostic imaging* ; Fracture Fixation, Internal/methods* ; Pelvic Bones/surgery* ; Metals ; Scapula/surgery* ; Models, Anatomic

In recent years, the study of single-cell transcriptome sequencing technology in the heterogeneity of astrocytes (astrocytes) after spinal cord injury (SCI) has provided new perspectives on post-traumatic nerve regeneration and repair. To provide a review on the research progress of single-cell sequencing technology in astrocytes after spinal cord injury (SCI), and to more comprehensively and deeply elaborate the application of single-cell sequencing technology in the field of astrocytes after SCI. Single-cell sequencing technology can analyse the transcriptomes of individual cells in a high-throughput manner, thus revealing fine differences in cell types and states. By using single-cell sequencing technology, the heterogeneity of astrocytes after SCI and their association with nerve regeneration and repair were revealed. In conclusion, the application of single-cell sequencing technology provides an important tool to reveal the heterogeneity of astrocytes after SCI, to further explore the mechanisms of astrocytes in SCI, and to develop intervention strategies targeting their regulatory mechanisms in order to improve the therapeutic efficacy of SCI. The discovery of changes in astrocyte transcriptome dynamics has improved researchers' understanding of spinal cord injury lesion progression and provided new insights into the treatment of spinal cord injury at different time points. To date, all of these findings need to be validated by more basic research and sufficient clinical trials. In the future, single-cell sequencing technology, through interdisciplinary collaboration with bioinformatics, computer science, tissue engineering, and clinical medicine, is expected to open a new window for the treatment of spinal cord injury.
Spinal Cord Injuries/metabolism* ; Astrocytes/cytology* ; Single-Cell Analysis/methods* ; Humans ; Animals ; Transcriptome ; Nerve Regeneration

OBJECTIVE: To explore clinical characteristics, lesion sites and correlation differences of different types of diabetic foot gangrene, and to provide evidence-based basis for clinical classification of diabetic foot gangrene. METHODS: A retrospective analysis was conducted on 266 patients with newly diagnosed diabetic foot gangrene who were admitted from January 2018 to December 2018, including 183 males and 83 females, aged from 35 to 92 years old with an average of (69.55±10.84) years old, and they were divided into wet gangrene group and dry gangrene group according to the different natures of gangrene. There were 139 patients in wet gangrene group, including 98 males and 41 females, aged from 35 to 90 years old with an average of (68.95±10.93) years old. There were 127 patients in dry gangrene group, including 85 males and 42 females, aged from 38 to 92 years old with an average of (70.21±10.75) years old. Body mass index (BMI), waist-to-hip ratio (WHR), body temperature, skin temperature difference between the affected and healthy sides of the lower extremities, and Wagner grade between two groups were recorded to evaluate symptoms and signs. The white blood cell count (WBC), neutrophil percentage (NEUT%), and C-reactive protein (C-reactive protein), erythrocyte sedimentation rate (ESR), procalcitonin (PCT), and interleukin-6 (IL-6) in peripheral blood between two groups were detected and compared to evaluate the infection status;the severity of diabetic peripheral neuropathy (DPN) was evaluated by using Toronto Clinical Scoring System (TCSS);the degree of pain in patients with diabetic foot gangrene was evaluated by numerical rating scale (NRS); ankle-brachial index (ABI) and popliteal artery blood flow velocity were used to evaluate the degree of arterial lesions. Spearman correlation analysis was used to analyze the correlations between gangrene TCSS, ABI and age, BMI, WHR, body temperature, calf skin temperature difference, WBC, NEUT%, CRP, ESR, PCT, IL-6, NRS, and Wagner classification indicators. RESULTS: The body temperature, skin temperature difference between the affected and healthy sides of the lower extremities, Wagner grade, WBC, NEUT%, CRP, ESR, PCT, IL-6, TCSS score, ABI, and popliteal artery blood flow velocity in wet gangrene group were higher than those in dry gangrene group (P<0.01), and BMI, WHR, and NRS score in dry gangrene group were higher than those in wet gangrene group;the differences were all statistically significant (P<0.01). The results of Spearman correlation analysis showed TCSS score of gangrene patients was correlated with body temperature (r=0.214), calf skin temperature difference (r=0.364), WBC (r=0.240), NEUT% (r=0.291), CRP (r=0.347), ESR (r=0.167), PCT (r=0.241), IL-6 (r=0.316), and popliteal fossa arterial blood flow velocity (r=0.261) and Wagner grade (r=0.273) were positively correlated, and the differences were statistically significant (P<0.01). ABI was negatively correlated with age (r=-0.183), BMI (r=-0.252), WHR (r=-0.288), and NRS score (r=-0.354), and the differences were statistically significant (P<0.01). CONCLUSION Diabetic foot gangrene is an extremely difficult and critical disease. Wet gangrene has a significant synergic effect with infection and neuropathy, while dry gangrene is closely related to vascular occlusion. The main contradiction of gangrene could be revealed through blood vessels, nerves and infection, providing evidence-based basis for the selection of debridement timing, anti-infection strategies and revascularization, with the aim of reducing the risk of amputation.
Humans ; Male ; Female ; Aged ; Middle Aged ; Diabetic Foot/diagnosis* ; Aged, 80 and over ; Adult ; Retrospective Studies ; Gangrene/physiopathology* ; C-Reactive Protein