Diterpenoid ferruginol is a key intermediate in biosynthesis of active ingredients such as tanshinone and carnosic acid.However, the traditional process of obtaining ferruginol from plants is often cumbersome and inefficient. In recent years, the increasingly developing gene editing technology has been gradually applied to the heterologous production of natural products, but the production of ferruginol in microbe is still very low, which has become an obstacle to the efficient biosynthesis of downstream chemicals, such as tanshinone. In this study, miltiradiene was produced by integrating the shortened diterpene synthase fusion protein,and the key genes in the MVA pathway were overexpressed to improve the yield of miltiradiene. Under the shake flask fermentation condition, the yield of miltiradiene reached about(113. 12±17. 4)mg·L~(-1). Subsequently, this study integrated the ferruginol synthase Sm CYP76AH1 and Sm CPR1 to reconstruct the ferruginol pathway and thereby realized the heterologous synthesis of ferruginol in Saccharomyces cerevisiae. The study selected the best ferruginol synthase(Il CYP76AH46) from different plants and optimized the expression of pathway genes through redox partner engineering to increase the yield of ferruginol. By increasing the copy number of diterpene synthase, CYP450, and CPR, the yield of ferruginol reached(370. 39± 21. 65) mg·L~(-1) in the shake flask, which was increased by 21. 57-fold compared with that when the initial ferruginol strain JMLT05 was used. Finally, 1 083. 51 mg·L~(-1) ferruginol was obtained by fed-batch fermentation, which is the highest yield of ferruginol from biosynthesis so far. This study provides not only research ideas for other metabolic engineering but also a platform for the construction of cell factories for downstream products.
Saccharomyces cerevisiae/genetics* ; Diterpenes/metabolism* ; Metabolic Engineering ; Fermentation ; Abietanes

Ganoderic acid is a class of lanostane-type triterpenoids found in Ganoderma species, and is one of the most important pharmacologically active components in G. lucidum, exhibiting antioxidant, anti-neuropsychiatric, anti-tumor, and immune-enhancing properties. The content of ganoderic acid in G. lucidum is very low, and the traditional extraction process is complex, yielding minimal amounts at high cost. The biosynthetic pathway of G. lucidum triterpenoids(GLTs), including the synthesis of different structural forms of ganoderic acid from lanosterol, as well as the molecular regulatory mechanisms involving key regulatory enzyme genes and their functions, are not yet fully understood. With the continuous development of synthetic biology technologies, there has been a deeper understanding of the biosynthesis and metabolic regulation pathways of ganoderic acid and its derivatives at the molecular level. Research has explored the key regulatory enzyme genes related to ganoderic acid biosynthesis and their functions. Moreover, through the optimization of synthetic biology and culture conditions, large-scale production and preparation of GLTs at the cellular level have been achieved. This paper reviews and analyzes the latest research progress on the biosynthesis pathways and metabolic regulation of GLTs, focusing on the configuration of ganoderic acid and its derivatives, the biosynthetic pathways, key enzyme genes, transcription factors related to ganoderic acid biosynthesis, signal transduction mechanisms, and factors affecting triterpenoid biotransformation. This review is expected to provide a theoretical basis and technical reference for improving the efficient production of triterpenoid pharmacological components and the exploitation and utilization of G. lucidum resources.
Triterpenes/chemistry* ; Reishi/chemistry* ; Biosynthetic Pathways ; Lanosterol

This study aims to explore the effects and action mechanisms of the active ingredients in Buyang Huanwu Decoction(BYHWD), namely tetramethylpyrazine(TMP) and hydroxy-safflor yellow A(HSYA), on oxygen-glucose deprivation/reglucose-reoxygenation(OGD/R)-induced inflammation and oxidative stress of microglia(MG). Network pharmacology was used to screen the effective monomer ingredients of BYHWD and determine the safe concentration range for each component. Inflammation and oxidative stress models were established to further screen the best ingredient combination and optimal concentration ratio with the most effective anti-inflammatory and antioxidant effects. OGD/R BV2 cell models were constructed, and BV2 cells in the logarithmic growth phase were divided into a normal group, a model group, an HSYA group, a TMP group, and an HSYA + TMP group. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of inflammatory cytokines such as interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and interleukin-6(IL-6). Oxidative stress markers, including superoxide dismutase(SOD), nitric oxide(NO), and malondialdehyde(MDA), were also measured. Western blot was used to analyze the protein expression of both inflammation-related pathway [Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)] and oxidative stress-related pathway [nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)]. Immunofluorescence was used to assess the expression of proteins such as inducible nitric oxide synthase(iNOS) and arginase-1(Arg-1). The most effective ingredients for anti-inflammatory and antioxidant effects in BYHWD were TMP and HSYA. Compared to the normal group, the model group showed significantly increased levels of IL-1β, TNF-α, IL-6, NO, and MDA, along with significantly higher protein expression of NF-κB, TLR4, Nrf2, and HO-1 and significantly lower SOD levels. The differences between the two groups were statistically significant. Compared to the model group, both the HSYA group and the TMP group showed significantly reduced levels of IL-1β, TNF-α, IL-6, NO, and MDA, lower expression of NF-κB and TLR4 proteins, higher levels of SOD, and significantly increased protein expression of Nrf2 and HO-1. Additionally, the expression of the M1-type MG marker iNOS was significantly reduced, while the expression of the M2-type MG marker Arg-1 was significantly increased. The results of the HSYA group and the TMP group had statistically significant differences from those of the model group. Compared to the HSYA group and the TMP group, the HSYA + TMP group showed further significant reductions in IL-1β, TNF-α, IL-6, NO, and MDA levels, along with significant reductions in NF-κB and TLR4 protein expression, an increase in SOD levels, and elevated Nrf2 and HO-1 protein expression. Additionally, the expression of the M1-type MG marker iNOS was reduced, while the M2-type MG marker Arg-1 expression increased significantly in the HSYA + TMP group compared to the TMP or HSYA group. The differences in the results were statistically significant between the HSYA + TMP group and the TMP or HSYA group. The findings indicated that the combined use of HSYA and TMP, the active ingredients of BYHWD, can effectively inhibit OGD/R-induced inflammation and oxidative stress of MG, showing superior effects compared to the individual use of either component.
Oxidative Stress/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Animals ; Mice ; Glucose/metabolism* ; Cell Line ; Inflammation/genetics* ; Oxygen/metabolism* ; Pyrazines/pharmacology* ; Microglia/metabolism* ; NF-E2-Related Factor 2/immunology* ; NF-kappa B/immunology* ; Toll-Like Receptor 4/immunology* ; Anti-Inflammatory Agents/pharmacology* ; Humans

OBJECTIVE: To examine the effectiveness of Chinese medicine (CM) Lianhua Qingwen Granule (LHQW) and Jingyin Gubiao Prescription (JYGB) in asymptomatic or mild patients with Omicron infection in the shelter hospital. METHODS: This single-center retrospective cohort study was conducted in the largest shelter hospital in Shanghai, China, from April 10, 2022 to May 30, 2022. A total of 56,244 asymptomatic and mild Omicron cases were included and divided into 4 groups, i.e., non-administration group (23,702 cases), LHQW group (11,576 cases), JYGB group (12,112 cases), and dual combination of LHQW and JYGB group (8,854 cases). The length of stay (LOS) in the hospital was used to assess the effectiveness of LHQW and JYGB treatment on Omicron infection. RESULTS: Patients aged 41-60 years, with nadir threshold cycle (C_T) value of N gene <25, or those fully vaccinated preferred to receive CM therapy. Before or after propensity score matching (PSM), the multiple linear regression showed that LHQW and JYGB treatment were independent influence factors of LOS (both P<0.001). After PSM, there were significant differences in LOS between the LHQW/JYGB combination and the other groups (P<0.01). The results of factorial design ANOVA proved that the LHQW/JYGB combination therapy synergistically shortened LOS (P=0.032). CONCLUSIONS Patients with a nadir C_T value <25 were more likely to accept CM. The LHQW/JYGB combination therapy could shorten the LOS of Omicron-infected individuals in an isolated environment.
Humans ; Drugs, Chinese Herbal/therapeutic use* ; Male ; Female ; Middle Aged ; Adult ; China/epidemiology* ; Hospitalization ; COVID-19 Drug Treatment ; COVID-19/epidemiology* ; SARS-CoV-2 ; Retrospective Studies ; Treatment Outcome ; Length of Stay ; Young Adult ; Aged

OBJECTIVE: To explore and quantify the association of hot night exposure during the sperm development period (0-90 lag days) with semen quality. METHODS: A total of 6,640 male sperm donors from 6 human sperm banks in China during 2014-2020 were recruited in this multicenter study. Two indices (i.e., hot night excess [HNE] and hot night duration [HND]) were used to estimate the heat intensity and duration during nighttime. Linear mixed models were used to examine the association between hot nights and semen quality parameters. RESULTS: The exposure-response relationship revealed that HNE and HND during 0-90 days before semen collection had a significantly inverse association with sperm motility. Specifically, a 1 °C increase in HNE was associated with decreased sperm progressive motility of 0.0090 (95% confidence interval [ CI]: -0.0147, -0.0033) and decreased total motility of 0.0094 (95% CI: -0.0160, -0.0029). HND was significantly associated with reduced sperm progressive motility and total motility of 0.0021 (95% CI: -0.0040, -0.0003) and 0.0023 (95% CI: -0.0043, -0.0002), respectively. Consistent results were observed at different temperature thresholds on hot nights. CONCLUSION Our findings highlight the need to mitigate nocturnal heat exposure during spermatogenesis to maintain optimal semen quality.
Humans ; Male ; Semen Analysis ; Adult ; Sperm Motility ; Hot Temperature/adverse effects* ; China ; Middle Aged ; Spermatozoa/physiology* ; Young Adult

Objective To investigate the effects of calorie-restricted diet(CRD)combined with intesti-nal micro-ecological preparation on the clinical outcomes of overweight/obese infertile patients undergoing fresh embryo transfer cycle.Methods A total of 197 cases of fresh embryo transfer assisted by in vitro fertili-zation(IVF)/intracytoplasmic sperm injection(ICSI)-embryo transfer(ET)in the hospital from January,2019 to December,2021 were selected as the research objects,and were divided into the overweight interven-tion group(n=46),the overweight control group(n=85)and the obesity intervention group(n=85)accord-ing to the body mass index(BMI)and whether they accepted CRD intervention or not.Results Compared to before the intervention,the overweight intervention group had significantly lower body mass[(63.59±4.90)kg vs.(67.47±5.10)kg],BMI[(25.74±1.26)kg/m2 vs.(26.58±0.75)kg/m2],body fat percentage[(36.51±3.76)％vs.(39.21±4.26)％],waist to hip ratio(0.88±0.04 vs.0.91±0.04),visceral fat grade(10.80±2.45 vs.12.63±2.60),and basal metabolic rate[(1 235.98±74.32)kcal vs.(1 254.63±77.23)kcal],P＜0.05;The body mass[(71.79±9.78)kg vs.(77.18±9.62)kg],BMI[(29.04±2.96)kg/m2 vs.(31.21±2.64)kg/m2],body fat percentage[(40.47±4.77)％vs.(43.97±3.81)％],waist to hip ratio(0.92±0.05 vs.0.96±0.06),and visceral fat grade(14.00±3.74 vs.16.59±3.15)of the obesity interven-tion group were significantly reduced(P＜0.05).Compared with the overweight control group,the number of transplanted embryos(1.85±0.36 vs.1.96±0.19)in the overweight intervention group was less(P＜0.05),while the number of live births(0.61±0.37 vs.0.56±0.36)was more,the live birth rate(43.48％vs.40.00％)was higher,but there was no statistically significant difference(P＞0.05).The clinical preg-nancy rate and live birth rate in the obesity intervention group were higher than those in the control group(72.41％vs.51.35％and 58.62％vs.35.14％),with no statistical significance,but with a strong statistical significance(P＞0.05).Conclusion CRD combined with intestinal micro-ecological preparation can signifi-cantly reduce the body mass and body fat of overweight/obese infertile patients,may help improve the success rate of assisted reproduction in obese infertile patients.

Objective To analyze the disease burden and change trend of breast cancer mortality among Chinese women from 2013 to 2021.Methods The indicators of breast cancer-related mortality (MR),age-standardized mortality rate (ASMR),deaths,and urban-rural deaths in the Chinese Cause of Death Surveil-lance Dataset from 2013 to 2021 were collected and collated,and the trend changes of MR,ASMR,and years lost in life of premature death (YLL) were analyzed,annual percentage change (APC) was used for trend a-nalysis,and Fisher substitution test was used to compare APC in different regions.Results From 2013 to 2021,a total of 88112 cases of breast cancer died,with an average annual MR of 7.56/100000 and an ASMR of 6.34/100000.From 2013 to 2021,the MR rate of breast cancer (APC=2.12%,t=5.414,P<0.001),MR in rural areas (APC=2.84%,t=7.581,P<0.001),and YLL in rural areas (APC=1.61%,t=3.588,P=0.009) showed an upward trend in China.MR at 40-<45 years old showed a downward trend (APC=-2.76%,t=-4.689,P=0.002),and at 70-<75 years old (APC=1.41%,t=2.494,P=0.041),75-<80 years old (APC=3.05%,t=3.690,P=0.008),and 80-<85 years old (APC=1.82%,t=2.909,P=0.023).There was a statistically significant difference in APC between MR and ASMR in urban and rural areas (P<0.001).There was no significant difference in the trend of other indicators (P>0.05).Conclusion From 2013 to 2021,the disease burden of breast cancer mortality in Chinese women showed an upward trend,and the comprehensive prevention and treatment of breast cancer should be strengthened to reduce the risk of breast cancer death.