The progress and promise of lineage reprogramming strategies for liver regeneration.

The liver exhibits remarkable regenerative capacity. However, the limited ability of primary human hepatocytes to proliferate in vitro, combined with a compromised regenerative capacity induced by pathological conditions in vivo, presents significant obstacles to effective liver regeneration following liver injuries and diseases. Developing strategies to compensate for the loss of endogenous hepatocytes is crucial for overcoming these challenges, and this remains an active area of investigation. Lineage reprogramming, the process of directly converting one cell type into another bypassing the intermediate pluripotent state, has emerged as a promising method for generating specific cell types for therapeutic purposes in regenerative medicine. Here, we discuss the recent progress and emergent technologies in lineage reprogramming into hepatic cells, and their potential applications in enhancing liver regeneration or treating liver disease models. We also address controversies and challenges that confront this field.

Region-specific cellular and molecular basis of liver regeneration after acute pericentral injury.

Liver injuries often occur in a zonated manner. However, detailed regenerative responses to such zonal injuries at cellular and molecular levels remain largely elusive. By using a fate-mapping strain, Cyp2e1-DreER, to elucidate liver regeneration after acute pericentral injury, we found that pericentral regeneration is primarily compensated by the expansion of remaining pericentral hepatocytes, and secondarily by expansion of periportal hepatocytes. Employing single-cell RNA sequencing, spatial transcriptomics, immunostaining, and in vivo functional assays, we demonstrated that the upregulated expression of the mTOR/4E-BP1 axis and lactate dehydrogenase A in hepatocytes contributes to pericentral regeneration, while activation of transforming growth factor ß (TGF-ß1) signaling in the damaged area mediates fibrotic responses and inhibits hepatocyte proliferation. Inhibiting the pericentral accumulation of monocytes and monocyte-derived macrophages through an Arg-Gly-Asp (RGD) peptide-based strategy attenuates these cell-derived TGF-ß1 signalings, thus improving pericentral regeneration. Our study provides integrated and high-resolution spatiotemporal insights into the cellular and molecular basis of pericentral regeneration.

Tuberculosis vaccine BCG: the magical effect of the old vaccine in the fight against the COVID-19 pandemic.

Bacillus Calmette-Guérin (BCG) is a live attenuated M. bovis vaccine that was developed about 100 years ago by Albert Calmette and Camille Guérin. Many countries have been using the vaccine for decades against tuberculosis (TB). The World Health Organization (WHO) recommends a single dose of BCG for infants in TB endemic as well as leprosy high risk countries, and globally almost 130 million infants are vaccinated yearly. The role of BCG is well known in reducing neonatal and childhood death rates. Epidemiological and retrospective cross-sectional studies demonstrated that the BCG vaccination protects the children against respiratory tract infections and lowers the risk of malaria in children. In addition, BCG enhances IFN-γ and IL-10 levels, thus providing immunity against respiratory tract infection even in elderly people. The BCG is also known to provide nonspecific innate immunity against viruses and parasites, through an innate immune mechanism termed 'trained immunity' and is defined as the immunological recall of the innate immune system by epigenetic reprogramming. Based on these studies it is suggested that the BCG has the potential to act as a protective agent against COVID-19. Further proven safety records of BCG in humans, its adjuvant activity and low-cost manufacturing make it an attractive option to stop the pandemic and reduce the COVID-19 related mortality. In this review we discuss the heterologous effects of BCG, induction of trained immunity and its implication in development of a potential vaccine against COVID-19 pandemic.

Parameter inversion and microscopic damage research on discrete element model of cement-stabilized steel slag based on 3D scanning technology.

The macro- and micro-physical properties of cement-stabilized steel slag (CSS) base materials in a highway project were studied. A discrete element model of CSS with a real steel slag shape was constructed using Particle Flow Code 3D and 3D scanning technology. The sensitivity between the macro- and micro-parameters of the sample was explored, and a nonlinear regression equation was established to analyze the relationship between these parameters. Uniaxial compression simulation tests were conducted on CSS with steel slag contents of 0%, 10%, 30%, and 50%. By combining contact calculation, crack location, energy tracking, acoustic emission (AE) monitoring, and other program systems, the macro- and micro-mechanical properties and micro-crack evolution law of the samples in the failure process were analyzed in terms of strength, energy, and fracture damage. The damage mechanism of CSS was also revealed. Results showed that with the increase in steel slag content, the elastic modulus and peak stress of the samples increased, the Poisson's ratio decreased, and the post-peak stress curve steepened, indicating obvious brittle failure characteristics. With the increase in steel slag content, the crack initiation stress, thickness of the fracture surface, and number of internal micro-cracks in CSS increased exponentially. In the uniaxial compression test, AE intensity underwent five stages, in which the peak moment of AE intensity exhibited hysteresis compared with the moment of the peak stress. Absorption and release phenomena of strain energy were observed in the process of specimen failure. When the steel slag content increased, the total strain energy absorbed by the specimen increased. When the absorbed energy exceeded the bond strength, the bond ruptured with the release of energy. The main crack of the sample penetrated and stretched to the direction of strain energy release to form a macroscopic fracture surface.

The Research Progress in Immunotherapy of Tuberculosis.

Tuberculosis (TB) is a serious public health problem worldwide. The combination of various anti-TB drugs is mainly used to treat TB in clinical practice. Despite the availability of effective antibiotics, effective treatment regimens still require long-term use of multiple drugs, leading to toxicity, low patient compliance, and the development of drug resistance. It has been confirmed that immune recognition, immune response, and immune regulation of Mycobacterium tuberculosis (Mtb) determine the occurrence, development, and outcome of diseases after Mtb infection. The research and development of TB-specific immunotherapy agents can effectively regulate the anti-TB immune response and provide a new approach toward the combined treatment of TB, thereby preventing and intervening in populations at high risk of TB infection. These immunotherapy agents will promote satisfactory progress in anti-TB treatment, achieving the goal of "ultra-short course chemotherapy." This review highlights the research progress in immunotherapy of TB, including immunoreactive substances, tuberculosis therapeutic vaccines, chemical agents, and cellular therapy.

Pilot Study of Blood Perfusion Changes at PC4 and Its Surrounding Points Induced by Acupuncture and Moxibustion.

Acupuncture and moxibustion are widely used in clinical practice; however, the differences between their mechanisms are unclear. In the present study, the response of blood perfusion resulting from acupuncture or moxibustion at Ximen (PC4) and its surrounding points was explored. Using the wavelet method, the differences in the frequency interval of blood flux were observed. Furthermore, the correlations between these points were analyzed. The results suggested that moxibustion could significantly improve blood flow perfusion at PC4 compared to acupuncture; however, there was no significant difference around PC4. The response of blood flux at PC4 to different stimulations was related to the frequency V (0.4-1.6 Hz) component. However, a difference in response at other points was not observed. Correlation analysis showed that both acupuncture and moxibustion could cause a decline in the correlation of blood flux signals at these recorded points, but there was no significant difference between these techniques. The results suggested that, at least in the forearm, the acupuncture or moxibustion only influenced the level of blood perfusion locally.

COVID-19 pandemic: SARS-CoV-2 specific vaccines and challenges, protection via BCG trained immunity, and clinical trials.

Introduction: The coronavirus disease 2019 (COVID-19) pandemic continues to spread worldwide and vaccination remains the most effective approach to control COVID-19. Currently, at least ten COVID-19 vaccines have been authorized under emergency authorization. However, these vaccines still face many challenges.Areas covered: This study reviews the concept and mechanisms of trained immunity induced by the Bacille Calmette Guérin (BCG) vaccine and identifies questions that should be answered before the BCG vaccine could be used to combat COVID-19 pandemic. Moreover, we present for the first time the details of current BCG vaccine clinical trials, which are underway in various countries, to assess its effectiveness in combating the COVID-19 pandemic. Finally, we discuss the challenges of COVID-19 vaccines and opportunities for the BCG vaccine. The literature was found by searching the PubMed (https://pubmed.ncbi.nlm.nih.gov/), Web of Science (www.webofknowledge.com), Embase (https://www.embase.com), and CNKI (https://www.cnki.net/) databases. The date was set as the default parameter for each database.Expert opinion: The advantages of the BCG vaccine can compensate for the shortcomings of other COVID-19 vaccines. If the efficacy of the BCG vaccine against COVID-19 is confirmed by these clinical trials, the BCG vaccine may be essential to resolve the challenges faced by COVID-19 vaccines.

Human primary epidermal organoids enable modeling of dermatophyte infections.

Technology of generating human epidermal derivatives with physiological relevance to in vivo epidermis is continuously investigated for improving their effects on modeling of human natural dermatological status in basic and clinical studies. Here, we report a method of robust establishment and expansion of human primary epidermal organoids (hPEOs) under a chemically defined condition. hPEOs reconstruct morphological, molecular, and functional features of human epidermis and can expand for 6 weeks. Remarkably, hPEOs are permissive for dermatophyte infections caused by Trichophyton Rubrum (T. rubrum). The T. rubrum infections on hPEOs reflect many aspects of known clinical pathological reactions and reveal that the repression on IL-1 signaling may contribute to chronic and recurrent infections with the slight inflammation caused by T. rubrum in human skin. Thus, our present study provides a new insight into the pathogenesis of T. rubrum infections and indicates that hPEOs are a potential ex vivo model for both basic studies of skin diseases and clinical studies of testing potential antifungal drugs.

Comprehensive proteomic atlas of skin biomatrix scaffolds reveals a supportive microenvironment for epidermal development.

Biomaterial scaffolds are increasingly being used to drive tissue regeneration. The limited success so far in human tissues rebuilding and therapy application may be due to inadequacy of the functionality biomaterial scaffold. We developed a new decellularized method to obtain complete anatomical skin biomatrix scaffold in situ with extracellular matrix (ECM) architecture preserved, in this study. We described a skin scaffold map by integrated proteomics and systematically analyzed the interaction between ECM proteins and epidermal cells in skin microenvironment on this basis. They were used to quantify structure and function of the skin's Matrisome, comprised of core ECM components and ECM-associated soluble signals that are key regulators of epidermal development. We especially revealed that ECM played a role in determining the fate of epidermal stem cells through hemidesmosome components. These concepts not only bring us a new understanding of the role of the skin ECM niche, they also provide an attractive combinational strategy based on tissue engineering principles with skin biomatrix scaffold materials for the acceleration and enhancement of tissue regeneration.

Enhanced differentiation of human pluripotent stem cells into pancreatic endocrine cells in 3D culture by inhibition of focal adhesion kinase.

BACKGROUND: Generation of insulin-producing cells from human pluripotent stem cells (hPSCs) in vitro would be useful for drug discovery and cell therapy in diabetes. Three-dimensional (3D) culture is important for the acquisition of mature insulin-producing cells from hPSCs, but the mechanism by which it promotes ß cell maturation is poorly understood. METHODS: We established a stepwise method to induce high-efficiency differentiation of human embryonic stem cells (hESCs) into mature monohormonal pancreatic endocrine cells (PECs), with the last maturation stage in 3D culture. To comprehensively compare two-dimensional (2D) and 3D cultures, we examined gene expression, pancreas-specific markers, and functional characteristics in 2D culture-induced PECs and 3D culture-induced PECs. The mechanisms were considered from the perspectives of cell-cell and cell-extracellular matrix interactions which are fundamentally different between 2D and 3D cultures. RESULTS: The expression of the pancreatic endocrine-specific transcription factors PDX1, NKX6.1, NGN3, ISL1, and PAX6 and the hormones INS, GCG, and SST was significantly increased in 3D culture-induced PECs. 3D culture yielded monohormonal endocrine cells, while 2D culture-induced PECs co-expressed INS and GCG or INS and SST or even expressed all three hormones. We found that focal adhesion kinase (FAK) phosphorylation was significantly downregulated in 3D culture-induced PECs, and treatment with the selective FAK inhibitor PF-228 improved the expression of ß cell-specific transcription factors in 2D culture-induced PECs. We further demonstrated that 3D culture may promote endocrine commitment by limiting FAK-dependent activation of the SMAD2/3 pathway. Moreover, the expression of the gap junction protein Connexin 36 was much higher in 3D culture-induced PECs than in 2D culture-induced PECs, and inhibition of the FAK pathway in 2D culture increased Connexin 36 expression. CONCLUSION: We developed a strategy to induce differentiation of monohormonal mature PECs from hPSCs and found limited FAK-dependent activation of the SMAD2/3 pathway and unregulated expression of Connexin 36 in 3D culture-induced PECs. This study has important implications for the generation of mature, functional ß cells for drug discovery and cell transplantation therapy for diabetes and sheds new light on the signaling events that regulate endocrine specification.

KIF9-AS1 promotes nasopharyngeal carcinoma progression by suppressing miR-16.

Long non-coding RNAs (lncRNAs) have been reported to serve a crucial role in the progression of nasopharyngeal carcinoma (NPC); however, the underlying molecular mechanisms of lncRNA KIF9-AS1 in the tumorigenesis of NPC remains poorly understood. Reverse transcription-quantitative PCR was used to analyze the expression levels of KIF9-AS1 and microRNA (miR)-16, and Cell Counting Kit-8, wound healing and Transwell assays were used to determine the cell viability, invasion and migration, respectively, of NPC cells. In addition, a dual-luciferase reporter assay was used to analyze the direct interaction between KIF9-AS1 and miR-16. NPC stage was classified according to the seventh edition of the AJCC staging system. The results revealed that KIF9-AS1 expression levels were upregulated in NPC tissues and cell lines. In addition, miR-16 was demonstrated to directly interact with KIF9-AS1 and inhibit KIF9-AS1 expression levels, whereas the miR-16 inhibitor rescued the effects of the KIF9-AS1-knockdown in NPC cells. Furthermore, the expression levels of KIF9-AS1 were upregulated, while those of miR-16 were downregulated in NPC tissues. Notably, the expression levels of KIF9-AS1 were observed to be significantly more upregulated in advanced tumors (III-IV vs. I-II) and patients with high KIF9-AS1 expression levels exhibited a worse prognosis. In conclusion, the findings of the present study suggested that KIF9-AS1 may promote the progression of NPC by targeting miR-16, thus KIF9-AS1 may be a novel molecular target for NPC therapy.

Long non-coding RNA HOXA-AS2 promotes the expression levels of hypoxia-inducible factor-1α and programmed death-ligand 1, and regulates nasopharyngeal carcinoma progression via miR-519.

Nasopharyngeal carcinoma (NPC) is a rare malignancy arising from the nasopharyngeal epithelium and belongs to the group of head and neck cancer types, which are usually associated with viral and/or environmental influences, as well as heredity causes. A recent study reported that the long non-coding RNA (lncRNA) HOXA cluster antisense RNA 2 (HOXA-AS2) may be a prognostic biomarker in NPC; however, the specific mechanisms underlying NCP progression are yet to be determined. The aim of the present study was to investigate the biological role of HOXA-AS2 in NPC. In the present study, the gene expression levels of HOXA-AS2, miR-519, hypoxia-inducible factor (HIF-1α) and programmed death-ligand 1 (PD-L1) were detected using reverse transcription-quantitative PCR (RT-qPCR) analysis and western blotting. Bioinformatics analysis and a dual luciferase reporter assay were performed to predict and confirm the direct interactions between HOXA-AS2 and microRNA (miR)-519, as well as between miR-519 and HIF-1α. A MTT assay was used to detect the cell viability, while cell migratory and invasive abilities were assessed using wound healing and Transwell assays. HOXA-AS2 and HIF-1α were found to be significantly upregulated in NPC tumor tissues, as well as in NPC cell lines. The overexpression of HOXA-AS2 significantly enhanced NPC progression, including the cell proliferative, migratory and invasive abilities. HOXA-AS2 was identified to directly bind to miR-519, whereas a miR-519 inhibitor significantly rescued the HOXA-AS2 knockdown-attenuated progression of NPC. Moreover, miR-519 could bind to HIF-1α and PD-L1. Overexpression of HIF-1α and PD-L1 significantly promoted NPC progression and partially recovered the phenotype of NPC cells attenuated by HOXA-AS2 knockdown. In conclusion, the present study demonstrated that HOXA-AS2/miR-519/HIF-1α and/or HOXA-AS2/miR-519/PD-L1 may be a novel mechanism regulating the progression of NPC, which may facilitate the development of targeted clinical therapy.

Author Correction: Connexin 32-mediated cell-cell communication is essential for hepatic differentiation from human embryonic stem cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Auto Micro Atomization Delivery of Human Epidermal Organoids Improves Therapeutic Effects for Skin Wound Healing.

Severe skin wounds are often associated with large areas of damaged tissue, resulting in substantial loss of fluids containing electrolytes and proteins. The net result is a vulnerability clinically to skin infections. Therapies aiming to close these large openings are effective in reducing the complications of severe skin wounds. Recently, cell transplantation therapy showed the potential for rapid re-epithelialization of severe skin wounds. Here, we show the improved effects of cell transplantation therapy using a robust protocol of efficient expansion and delivery of epidermal cells for treatment of severe skin wounds. Human skin tissues were used to generate human epidermal organoids maintained under newly established culture conditions. The human epidermal organoids showed an improved capacity of passaging for at least 10 rounds, enabling organoids to expand to cell numbers required for clinical applications. A newly designed auto micro-atomization device (AMAD) was developed for delivery of human epidermal organoids onto the sites of severe skin wounds enhancing uniform and concentrated delivery of organoids, facilitating their engraftment and differentiation for skin reconstitution. With the optimal design and using pneumatic AMAD, both survival and functions of organoids were effectively protected during the spraying process. Cells in the sprayed human epidermal organoids participated in the regeneration of the epidermis at wound sites in a mouse model and accelerated wound healing significantly. The novel AMAD and out new protocol with enhanced effects with respect to both organoid expansion and efficient transplantation will be used for clincal treatments of complex, uneven, or large-area severe skin wounds.

Human ESC-derived expandable hepatic organoids enable therapeutic liver repopulation and pathophysiological modeling of alcoholic liver injury.

We report the generation of human ESC-derived, expandable hepatic organoids (hEHOs) using our newly established method with wholly defined (serum-free, feeder free) media. The hEHOs stably maintain phenotypic features of bipotential liver stem/progenitor cells that can differentiate into functional hepatocytes or cholangiocytes. The hEHOs can expand for 20 passages enabling large scale expansion to cell numbers requisite for industry or clinical programs. The cells from hEHOs display remarkable repopulation capacity in injured livers of FRG mice following transplantation, and they differentiate in vivo into mature hepatocytes. If implanted into the epididymal fat pads of immune-deficient mice, they do not generate non-hepatic lineages and have no tendency to form teratomas. We further develop a derivative model by incorporating human fetal liver mesenchymal cells (hFLMCs) into the hEHOs, referred to as hFLMC/hEHO, which can model alcoholic liver disease-associated pathophysiologic changes, including oxidative stress generation, steatosis, inflammatory mediators release and fibrosis, under ethanol treatment. Our work demonstrates that the hEHOs have considerable potential to be a novel, ex vivo pathophysiological model for studying alcoholic liver disease as well as a promising cellular source for treating human liver diseases.

Sweat gland organoids contribute to cutaneous wound healing and sweat gland regeneration.

Sweat glands perform a vital thermoregulatory function in mammals. Like other skin components, they originate from epidermal progenitors. However, they have low regenerative potential in response to injury. We have established a sweat gland culture and expansion method using 3D organoids cultures. The epithelial cells derived from sweat glands in dermis of adult mouse paw pads were embedded into Matrigel and formed sweat gland organoids (SGOs). These organoids maintained remarkable stem cell features and demonstrated differentiation capacity to give rise to either sweat gland cells (SGCs) or epidermal cells. Moreover, the bipotent SGO-derived cells could be induced into stratified epidermis structures at the air-liquid interface culture in a medium tailored for skin epidermal cells in vitro. The SGCs embedded in Matrigel tailored for sweat glands formed epithelial organoids, which expressed sweat-gland-specific markers, such as cytokeratin (CK) 18 and CK19, aquaporin (AQP) 5 and αATP. More importantly, they had potential of regeneration of epidermis and sweat gland when they were transplanted into the mouse back wound and claw pad with sweat gland injury, respectively. In summary, we established and optimized culture conditions for effective generation of mouse SGOs. These cells are candidates to restore impaired sweat gland tissue as well as to improve cutaneous skin regeneration.

Connexin 32 and connexin 43 are involved in lineage restriction of hepatic progenitor cells to hepatocytes.

BACKGROUND: Bi-potential hepatic progenitor cells can give rise to both hepatocytes and cholangiocytes, which is the last phase and critical juncture in terms of sequentially hepatic lineage restriction from any kind of stem cells. If their differentiation can be controlled, it might access to functional hepatocytes to develop pharmaceutical and biotechnology industries as well as cell therapies for end-stage liver diseases. METHODS: In this study, we investigated the influence of Cx32 and Cx43 on hepatocyte differentiation of WB-F344 cells by in vitro gain and loss of function analyses. An inhibitor of Cx32 was also used to make further clarification. To reveal p38 MAPK pathway is closely related to Cxs, rats with 70% partial hepatectomy were injected intraperitoneally with a p38 inhibitor, SB203580. Besides, the effects of p38 MAPK pathway on differentiation of hepatoblasts isolated from fetal rat livers were evaluated by addition of SB203580 in culture medium. RESULTS: In vitro gain and loss of function analyses showed overexpression of Connexin 32 and knockdown of Connexin 43 promoted hepatocytes differentiation from hepatic progenitor cells. In addition, in vitro and ex vivo research revealed inhibition of p38 mitogen-activated protein kinase pathway can improve hepatocytes differentiation correlating with upregulation of Connexin 32 expression and downregulation of Connexin 43 expression. CONCLUSIONS: Here we demonstrate that Connexins play crucial roles in facilitating differentiation of hepatic progenitors. Our work further implicates that regulators of Connexins and their related pathways might provide new insights to improve lineage restriction of stem cells to mature hepatocytes.

[Impact of Biodegradable Organic Matter on the Functional Microbe Activities in Partial Nitrification Granules].

To explore the short-term impact of biodegradable organic matter on the activities of different functional microbes in autotrophic partial nitrification granular sludge (PNG),the variations of both nitrogen transformation performance and dissolved oxygen (DO) uptake of PNG were investigated in this study,by carrying out successive batch tests with and without the organics stressing.The results showed that the higher the C/N ratio,the lower the specific nitrite accumulation rate of q(NO2--N).Meanwhile,the increase of heterotrophic bacteria (HeB) activities caused the fast DO uptake by PNG,which could effectively suppress nitrite oxidizing bacteria (NOB) with the low oxygen affinity.When inorganic substrate culture was employed in the following phase,both HeB and NOB showed low activities,with significant increase in q(NO2--N).In short,the adverse effects of biodegradable organic matter on the performance of PNG system were partially reversible,which could benefit to enhance the advantage of ammonium oxidizing bacteria (AOB) and improve the stability of partial nitrification reaction.

Connexin 32-mediated cell-cell communication is essential for hepatic differentiation from human embryonic stem cells.

Gap junction-mediated cell-cell interactions are highly conserved and play essential roles in cell survival, proliferation, differentiation and patterning. We report that Connexin 32 (Cx32)-mediated gap junctional intercellular communication (GJIC) is necessary for human embryonic stem cell-derived hepatocytes (hESC-Heps) during step-wise hepatic lineage restriction and maturation. Vitamin K2, previously shown to promote Cx32 expression in mature hepatocytes, up-regulated Cx32 expression and GJIC activation during hepatic differentiation and maturation, resulting in significant increases of hepatic markers expression and hepatocyte functions. In contrast, negative Cx32 regulator 2-aminoethoxydiphenyl borate blocked hESC-to-hepatocyte maturation and muted hepatocyte functions through disruption of GJIC activities. Dynamic gap junction organization and internalization are phosphorylation-dependent and the p38 mitogen-activated protein kinases pathway (MAPK) can negatively regulate Cxs through phosphorylation-dependent degradation of Cxs. We found that p38 MAPK inhibitor SB203580 improved maturation of hESC-Heps correlating with up-regulation of Cx32; by contrast, the p38 MAPK activator, anisomycin, blocked hESC-Heps maturation correlating with down-regulation of Cx32. These results suggested that Cx32 is essential for cell-cell interactions that facilitate driving hESCs through hepatic-lineage maturation. Regulators of both Cx32 and other members of its pathways maybe used as a promising approach on regulating hepatic lineage restriction of pluripotent stem cells and optimizing their functional maturation.

Study on the Fire Damage Characteristics of the New Qidaoliang Highway Tunnel: Field Investigation with Computational Fluid Dynamics (CFD) Back Analysis.

In the New Qidaoliang Tunnel (China), a rear-end collision of two tanker trunks caused a fire. To understand the damage characteristics of the tunnel lining structure, in situ investigation was performed. The results show that the fire in the tunnel induced spallation of tunnel lining concrete covering 856 m³; the length of road surface damage reached 650 m; the sectional area had a maximum 4% increase, and the mechanical and electrical facilities were severely damaged. The maximum area loss happened at the fire spot with maximum observed concrete spallation up to a thickness of 35.4 cm. The strength of vault and side wall concrete near the fire source was significantly reduced. The loss of concrete strength of the side wall near the inner surface of tunnel was larger than that near the surrounding rock. In order to perform back analysis of the effect of thermal load on lining structure, simplified numerical simulation using computational fluid dynamics (CFD) was also performed, repeating the fire scenario. The simulated results showed that from the fire breaking out to the point of becoming steady, the tunnel experienced processes of small-scale warming, swirl around fire, backflow, and longitudinal turbulent flow. The influence range of the tunnel internal temperature on the longitudinal downstream was far greater than on the upstream, while the high temperature upstream and downstream of the transverse fire source mainly centered on the vault or the higher vault waist. The temperature of each part of the tunnel near the fire source had no obvious stratification phenomenon. The temperature of the vault lining upstream and downstream near the fire source was the highest. The numerical simulation is found to be in good agreement with the field observations.