Objectives: Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) defi ciency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) ofPTH through in vitro functional study. Methods: Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbu tyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Results: Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified.Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%–38%, and 51%–96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. Conclusions In this study, two novel RVs of PTH(Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

Objectives: Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) defi ciency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) ofPTH through in vitro functional study. Methods: Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbu tyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Results: Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified.Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%–38%, and 51%–96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. Conclusions In this study, two novel RVs of PTH(Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

Objectives: Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) defi ciency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) ofPTH through in vitro functional study. Methods: Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbu tyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Results: Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified.Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%–38%, and 51%–96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. Conclusions In this study, two novel RVs of PTH(Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

Objectives: Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) defi ciency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) ofPTH through in vitro functional study. Methods: Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbu tyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Results: Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified.Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%–38%, and 51%–96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. Conclusions In this study, two novel RVs of PTH(Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

Objectives: Hypoparathyroidism (HP) is a rare endocrine disorder caused by parathyroid hormone (PTH) defi ciency. The PTH is a candidate gene for familial isolated hypoparathyroidism (FIH). This study aimed to investigate the pathogenicity of two novel rare variants (RVs) ofPTH through in vitro functional study. Methods: Targeted next-generation sequencing was used to identify candidate gene mutations. Clinical data were retrospectively collected. Wild-type (WT) PTH was used as a template for site-directed mutagenesis to create mutant eukaryotic expression plasmids, which were transfected into cells. Treated with or without 4-phenylbu tyric acid (4-PBA), the levels of intact PTH (iPTH) and PTH (1-84) were measured by chemiluminescence, and protein expression was assessed using Western blotting. Results: Two patients carrying PTH mutations (c.154G > A: p.Val52Ile, c.270G > T: p.Leu90Phe) were identified.Patient 1, a 45-year-old male, presented with carpal and pedal numbness, muscle cramps, and low serum calcium (1.29 mmol/L). Patient 2, a 12-year-old female, had muscle twitches, convulsions, low calcium (1.50 mmol/L), and iPTH of 4 pg/mL. The iPTH or PTH (1-84) levels in the medium transfected with mutant Val52Ile and Leu90Phe PTH decreased by 31%–38%, and 51%–96% compared to WT (allP < 0.05), which were not rescued by 4-PBA. No significant changes in intracellular PTH expression were observed. Conclusions In this study, two novel RVs of PTH(Val52Ile and Leu90Phe) were identified that may impair hormone synthesis and secretion. Our study has broadened the mutation spectrum of the PTH and shed light on potential mechanisms underlying FIH.

Objective To explore the effects of Shuanglu Tongnao Formula on neuroinflammation in ischemic stroke (IS) rats via the P2X purinoceptor 7 receptor (P2X7R)/NLR family pyrin domain-containing 3 (NLRP3) pathway. Methods The rats were divided into five groups: the IS group, control group, Shuanglu Tongnao Formula group, P2X7R inhibitor brilliant blue G (BBG) group, and Shuanglu Tongnao Formula combined with P2X7R activator adenosine triphosphate (ATP) group, with 18 rats in each group. Except for the control group, rats in all other groups were used to construct an IS model using the suture method. After successful modeling, the drug was given once a day for 2 weeks. Neurological function scores and cerebral infarction volume ratios were measured in rats. Pathological examination of the ischemic penumbra brain tissue was performed. Immunofluorescence staining was used to quantify the proportions of microglia co-expressing both inducible nitric oxide synthase (iNOS) and ionized calcium-binding adapter molecule 1 (Iba1), as well as arginase 1 (Arg1) and Iba1, in the ischemic penumbra brain tissue. ELISA was used to detect tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β), interleukin 6 (IL-6) and IL-10 in the ischemic penumbra brain tissue. Western blotting was used to measure P2X7R, NLRP3, and IL-1β proteins in the ischemic penumbra brain tissue. Results Compared with the control group, the IS group showed disordered neuronal arrangement, nuclear condensation, and obvious infiltration of inflammatory cells in the ischemic penumbra; significantly elevated neurological function scores, cerebral infarction volume ratios, proportions of microglia co-expressing iNOS and Iba1, and levels of TNF-α, IL-6, and P2X7R, NLRP3, IL-1β proteins; along with reduced proportions of microglia co-expressing Arg1 and Iba1 and levels of TGF-β and IL-10. Compared with the IS group, the Zhuang medicine Shuanglu Tongnao Formula and BBG groups demonstrated alleviated brain tissue damage; reduced neurological function scores, cerebral infarction volume ratios, proportions of microglia co-expressing iNOS and Iba1, and levels of TNF-α, IL-6, and P2X7R, NLRP3, IL-1β proteins; along with increased proportions of microglia co-expressing Arg1 and Iba1 and levels of TGF-β and IL-10. ATP reversed the effects of Zhuang medicine Shuanglu Tongnao Formula on microglial polarization and neuroinflammation in IS rats. Conclusion Zhuang medicine Shuanglu Tongnao Formula may promote the transformation of microglia from M1 type to M2 type by inhibiting the P2X7R/NLRP3 pathway, thereby improving neuroinflammation in IS rats.
Animals ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Receptors, Purinergic P2X7/metabolism* ; Male ; Drugs, Chinese Herbal/pharmacology* ; Rats ; Ischemic Stroke/pathology* ; Rats, Sprague-Dawley ; Disease Models, Animal ; Signal Transduction/drug effects* ; Neuroinflammatory Diseases/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Nitric Oxide Synthase Type II/metabolism* ; Interleukin-10/metabolism* ; Brain Ischemia/drug therapy* ; Microglia/metabolism*

Objective To explore the effect of Shuanglu Tongnao Formula on neuronal ferroptosis in ischemic stroke rats and its regulatory mechanism on the silent information regulator 2 homolog 1(SIRT1)/nuclear factor erythroid 2-related fac-tor 2(Nrf2)/glutathione peroxidase 4(GPx4)signaling pathways.Methods Twenty rats were selected as sham operation group by the random number table method,and the remaining seventy rats were made ischemic stroke rat models by the middle cerebral artery occlusion method.The rats that had been successfully modeled were randomly divided into the model control group,Shuanglu Tongnao formula group,Shuanglu Tongnao formula+SIRT1 inhibitor group(Shuanglu Tongnao formula+EX527 group),with 20 rats in each group.After 14 days,the rats were scored for neurological injury;TTC staining was applied to detect the area of cerebral infarction in rats;HE staining was applied to detect pathological changes in rat brain tissue;Nissl staining was applied to detect the number of neurons in rat brain tissue;the kit was applied to detect the levels of ferri ion(Fe2+),superoxide dismutase(SOD),glutathione(GSH),and malonaldehyde(MDA)in rat brain tissue;immunohistochemistry was applied to de-tect the positive expression of acyl-CoA synthetase long-chain family member 4(ACSL4),transferrin receptor(TFR),and ferritin heavy polypeptide 1(FTH1)proteins in rat brain tissue;Western blotting method was applied to detect the expression of SIRT1,Nrf2,GPx4,and cystine/glutamate antiporter solute carrier family 7 member 11(SLC7A11)proteins in rat brain tissue.Results Compared with the sham operation group,the neurological deficit score,cerebral infarction area,the contents of Fe2+and MDA,and the protein expressions of ACSL4 and TFR in model control group were increased(P<0.05);the number of neurons,the con-tents of SOD and GSH,the protein expression of FTH1,SIRT1,Nrf2,GPx4,and SLC7A11 were all reduced(P<0.05).Compared with the model control group,the neurological deficit score,cerebral infarction area,the contents of Fe2+and MDA,and the protein expression of ACSL4 and TFR in the Shuanglu Tongnao formula group were reduced(P<0.05),and the number of neurons,the contents of SOD and GSH,the protein expressions of FTH1,SIRT1,Nrf2,GPx4,and SLC7A11 are all increased(P<0.05).The results of the SIRT1 inhibitor supplementation experiment showed that the SIRT1 inhibitor reversed the inhibitory effect of Shuan-glu Tongnao formula on neuronal ferroptosis,while also inhibited the expression of Nrf2 and GPx4(P<0.05).Conclusion The Shuanglu Tongnao formula may inhibit neuronal ferroptosis in ischemic stroke rats by activating the SIRT1/Nrf2/GPx4 signa-ling pathway.

ObjectiveTo quantitatively investigate the changes in the total volume and contour density of hepatic oval cells （HOC） in hepatic lobules of rats with carbon tetrachloride （CCl₄）-induced hepatic fibrosis. MethodsA total of 11 healthy male Sprague-Dawley rats were randomly divided into control group with 5 rats and hepatic fibrosis group with 6 rats， and CCl₄ and olive oil suspension were injected subcutaneously twice a week， 3 mL/kg each time. After five weeks of hepatic fibrosis modeling， five liver tissue blocks with a size of about 1 mm³ were randomly selected from the liver of each rat to prepare one Epon812 epoxy resin-embedded ultrathin section， and the stereological method and transmission electron microscopy were used for the quantitative analysis of the total volume and contour density of HOC in the hepatic lobules of rats. In addition， four liver tissue blocks with a thickness of 2 mm were randomly selected from the remaining liver of each rat to prepare two paraffin-embedded Masson staining sections， and the degree of liver fibrosis in each rat was qualitatively evaluated according to the Metavir staging criteria for liver fibrosis. The independent-samples t test was used for comparison of continuous data between groups. ResultsThe quantitative stereological analysis showed that the total volume of HOC in hepatic lobules was 15.40±7.63 mm³ in the control group and 146.80±114.00 mm³ in the liver fibrosis group， and compared with the control group， the total volume of HOC in hepatic lobules of rats in the liver fibrosis group was significantly increased by 8.53 times （t=-2.551， P=0.031）； the contour density of HOC in hepatic lobules was 56.20±40.40 in the control group and 566.50±317.00 in the liver fibrosis group， and compared with the control group， the contour density of HOC in hepatic lobules of rats in the liver fibrosis group was significantly increased by 9.08 times （t=-3.539， P=0.006）. Qualitative observation showed that liver fibrosis stage of rats reached stage Ⅱ-Ⅲ according to the Metavir scoring criteria， and massive proliferation of HOC was observed around the proliferation site of hepatic stellate cells in the perisinusoidal space of rats. ConclusionCCl₄ induces significant proliferation of HOC in hepatic lobules of rats with liver fibrosis.

Objective To explore the mechanism of Zhuangxuan Yin in the treatment of children with H1N1 pneumonia through regulating gut microbiota mediated by p38 mitogen-activated protein kinase(p38-MAPK)pathway.Methods A BALB/c mouse model of H1N1 pneumonia was prepared using the H1N1 influenza virus allantoic solution for nasal drop.The model was randomly separated into 5 groups:model group,Zhuangxuan Yin low-,medium-and high-dose groups,and high-dose Zhuangxuan Yin(28.66 g·kg-1)+anisomycin(10 mg·kg-1)group.The control group was infused with sterile physiological saline of equal volume using the same method.After treatment with Zhuangxuan Yin and anisomycin,the lung index of mice in each group was measured,and HE staining was applied to detect the pathological morphology of lung and large intestine tissues.16SrRNA gene sequencing was applied to detect the structural difference of gut microbiota in mice of each group.Enzyme-linked immunosorbent assay(ELASA)was applied to measure the levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-4,IL-6,and IL-1β in bronchoalveolar lavage fluid(BALF)and large intestine tissue of mice in each group.Western Blot was applied to detect the expression of p38-MAPK pathway-related proteins in lung and large intestine tissues of mice in each group.Results Compared with the control group,the lung and large intestine tissues of the model group mice showed obvious pathological damage,the lung index,pathological score of lung and large intestine,ACE index,Shannon index,abundance of class Clostridia,the levels of TNF-α,IL-4,IL-6,and IL-1β in BALF and large intestine tissues,and p-p38-MAPK/p38-MAPK in lung and large intestine tissues increased(P＜0.05).The abundance of class Bacteroidales decreased(P＜0.05).Compared with the model group,the pathological damage in the lung and large intestine tissues of mice in the Zhuangxuan Yin low-,medium-and high-dose groups were reduced.The lung index,pathological score of lung and large intestine,ACE index,Shannon index,abundance of class Clostridia,the levels of TNF-α,IL-4,IL-6,and IL-1β in BALF and large intestine tissues,and p-p38-MAPK/p38-MAPK in lung and large intestine tissues decreased(P＜0.05),the abundance of class Bacteroidales increased(P＜0.05),and in a dose-dependent manner(P＜0.05).Compared with the high-dose Zhuangxuan Yin group,the pathological damage in the lung and large intestine tissues of mice in the high-dose Zhuangxuan Yin+anisomycin group was aggravated,the lung index,pathological score of lung and large intestine,ACE index,Shannon index,abundance of class Clostridia,the levels of TNF-α,IL-4,IL-6,and IL-1β in BALF and large intestine tissues,and p-p38-MAPK/p38-MAPK in lung and large intestine tissues increased(P＜0.05),and the abundance of class Bacteroidales decreased(P＜0.05).Conclusion Zhuangxuan Yin can improve the imbalance of intestinal microbiota in H1N1 pneumonia mice by inhibiting p38-MAPK signal activation,thereby inhibiting inflammation and reducing lung and large intestine tissue damage in mice,which may have a therapeutic effect on children with H1N1 pneumonia.

Objective To investigate the expression levels of HBV pregenomic RNA (pgRNA) and hepatitis B core-related antigen (HBcrAg) in circulating serum of chronic hepatitis B (CHB) patients after withdrawal from nucleos(t)ide analogues (NUC), as well as the correlation of HBV pgRNA and HBcrAg levels in circulating blood in different periods of time with recurrence in CHB patients after drug withdrawal. Methods Among the patients who attended the outpatient service of Affiliated Hospital of North Sichuan Medical College from December 2019 to July 2022, a total of 108 CHB patients who received anti-HBV therapy for at least 5 years and met the criteria for drug withdrawal in 2017 EASL Guidelines were enrolled. According to the time of drug withdrawal, the patients were divided into 4-, 12-, and 24-week groups after drug withdrawal, and according to the presence or absence of recurrence, they were divided into recurrence group and non-recurrence group. Quantitative real-time PCR was used to measure the level of HBV pgRNA in circulating serum of CHB patients; ELISA was used to measure the expression level of HBcrAg in peripheral venous blood; quantitative real-time PCR was used to measure HBV DNA load with high accuracy. The t -test was used for comparison of continuous data between two groups. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t -test was used for further comparison between two groups. The Pearson correlation test was used to investigate the correlation between the indices in circulating blood. Results For the CHB patients after drug withdrawal, the recurrence rate was 17.1% at 4-12 weeks, cumulative recurrence rate reached 29.3% after 24 weeks of follow-up, the patients with positive HBV DNA alone accounted for 64.3% and 60.0%, respectively, those with positive HBeAg alone accounted for 28.5% and 20.0%, respectively, and those with positive HBV DNA and HBeAg accounted for 7.1% and 20.0%, respectively. The expression levels of HBV pgRNA, HBcrAg, and HBV DNA in circulating serum of CHB patients at 24 weeks after drug withdrawal were significantly higher than those at the time of drug withdrawal and at 4 weeks after drug withdrawal, and there was a significant difference between groups at different time points (all P < 0.05). Compared with the non-recurrence group, the recurrence group had significantly higher expression levels of HBV pgRNA, HBcrAg, and HBV DNA in circulating serum ( t =2.549, 8.654, and 27.429, all P < 0.05), and further analysis of the recurrence group showed that the levels of HBV pgRNA, HBcrAg, and HBV DNA in circulating serum at 12-24 weeks were significantly higher than those at 4-12 weeks (all P < 0.05). At the time of drug withdrawal, the recurrence group had significantly higher expression levels of HBV pgRNA and HBcrAg in circulating serum than the non-recurrence group ( t =18.561 and 6.152, both P < 0.001). The Pearson correlation analysis showed that in the recurrence group after drug withdrawal, HBV pgRNA and HBcrAg were positively correlated with HBV DNA in circulating serum ( r =0.82 and 0.66, both P < 0.001), while no such correlation was observed in the non-recurrence group ( r =0.14 and 0.04, both P > 0.05). Conclusion The recurrence group had significantly higher expression levels of HBV pgRNA and HBcrAg than the non-recurrence group at the time of drug withdrawal, suggesting that the levels of HBV pgRNA and HBcrAg in the CHB patients of the non-recurrence group at the time of drug withdrawal may be used as the reference thresholds for safe drug withdrawal in CHB patients, and measurement of HBV pgRNA and HBcrAg may be one of the potential reference indicators for the selection of anti-HBV treatment endpoints in the future.